The objective of this study was to determine the effect of heat shock (HS) on the Ca(2+) release and the subsequent development in matured porcine oocytes. Oocytes were matured in vitro and randomly allocated to different heat treatments at 41.5 degrees C for 1 (HS1h), 2 (HS2h) or 4h (HS4h). Control groups of oocytes were cultured for 0 or 4h without HS (39 degrees C, C0h, C4h). In Experiment 1 (eight replicates), matured oocytes were activated by thimerosal (200 microM, 10 min) following HS. Among all heated groups, maximal intracellular calcium concentration ([Ca(2+)](i)) was the highest in the HS2h. The lowest [Ca(2+)](i) peak among HS groups was observed in the HS4h, but it was higher than that in the non-heated C4h group (P<0.05). In Experiment 2 (12 replicates), each matured oocyte was injected with IP(3) (0.5mM) and the Ca(2+) transient was recorded. The peak [Ca(2+)](i) in the C4h group was still the lowest among all groups (P<0.05). Total Ca(2+) release in HS2h appeared the highest among all treatments, and it was significantly higher than that in HS1h and C4h groups (P<0.05). In order to clarify the effect of incubation time in vitro (Experiment 3), matured oocytes were cultured at 39 degrees C for 0, 2 and 4h prior to treatment with thimerosal or injected with IP(3) (three replicates). The Ca(2+) release of matured oocytes declined with the prolonged culture (P<0.05). Finally, the development of HS-oocytes was evaluated after parthenogenetic activation (Experiment 4, three replicates), and the proportion of embryos developing to the blastocysts were lower (P<0.05) in the HS groups (31+/-7% to 33+/-1%) than in the control groups (52+/-11% to 56+/-9%). We conclude that HS alters the Ca(2+)-releasing ability of matured pig oocytes, and that heat-shocked oocytes with greater Ca(2+) release incur a low developmental competence after parthenogenetic activation.
Inositol 1,4,5-trisphosphate (IP3) is considered to be important for activation of mammalian oocytes at the time of fertilization, and activation induces a rise in intracellular Ca2+ concentration ([Ca2+]i) by release from the Ca2+ stores in the oocytes. Therefore, IP3 could act as an artificial activator of porcine oocytes. Activation and development, and rise in [Ca2+]i in matured oocytes injected with various concentrations of IP3 were investigated in this study. Porcine oocytes were recovered from the ovaries of prepubertal gilts, matured for 46-48 h and cultured in vitro for 7 days in following treatments as non-injected oocytes (NI), injected with carrier buffer, 2.5, 5 and 500 microM of IP3. The result showed that IP3 activated porcine oocytes matured in vitro (NI 3.8%, buffer 7.1%, 2.5 microM IP3 73.5%, 5 microM IP3 76.2%, 500 microM IP3 85.2%). There was a slight but not significant increase in the proportion of oocytes activated as the level of IP3 increased. The rate of development to the cleavage stage increased remarkably when the concentration of IP3 increased (NI 4.9%, buffer 5.7%, 2.5 microM IP3 30.3%, 5 microM IP3 47.1%, 500 microM IP3 78.1%). Blastocyst development was only observed in oocytes that had been injected with a higher concentration of IP3 (5 microM IP3 6.1% and 500 microM IP3 5.3%). Both the peak value and duration of [Ca2+]i rise also increased as the concentration of IP3 increased. Baseline values (ratio value, R) for [Ca2+]i ranged from 1.51 to 1.57 and was not affected by the buffer treatment. The peak value of [Ca2+]i rose significantly with increasing level of IP3 treatment (2.5 microM IP3, 3.54 +/- 0.32; 5 microM IP3, 7.50 +/- 0.37; 500 microM IP3, 8.54 +/- 0.33). Similarly, the duration of the [Ca2+]i rise increased as the level of IP3 increased (2.5 microM IP3, 43.7+/- 7.00 s; 5 microM IP3, 93.5 +/- 9.17 s; 500 microM IP3, 160.6 +/- 18.9 s). It was concluded that injected IP3 promotes the development of porcine matured oocytes and that their developmental ability is positively correlated with the rise in [Ca2+]i induced by IP3.
The objectives of this study included: (1) identify the expression of miRNAs specific to bovine cumulus-oocyte complexes (COCs) during late oogenesis, (2) characterize the expression of candidate miRNAs as well as some miRNA processing genes, and (3) computationally identify and characterize the expression of target mRNAs for candidate miRNAs. Small RNAs in the 16-27 bp range were isolated from pooled COCs aspirated from 1- to 10-mm follicles of beef cattle ovaries and used to construct a cDNA library. A total 1798 putative miRNA sequences from the cDNA library of small RNA were compared to known miRNAs. Sixty-four miRNA clusters matched previously reported sequences in the miRBase database and 5 miRNA clusters had not been reported. TaqMan miRNA assays were used to confirm the expression of let-7b, let-7i, and miR-106a from independent collections of COCs. Real-time PCR assays were used to characterize expression of miRNA processing genes and target mRNAs (MYC and WEE1A) for the candidate miRNAs from independent collections of COCs. Expression data were analyzed using general linear model procedures for analysis of variance. The expression of let-7b and let-7i were not different between the cellular populations from various sized follicles. However, miR-106a expression was greater (P<0.01) in oocytes compared with COCs and granulosa cells. Furthermore, all the miRNA processing genes have greater expression (P<0.001) in oocytes compared with COCs and granulosa cells. The expression of potential target mRNAs for let-7 and let-7i (i.e., MYC), and miR-106a (i.e., WEE1A) were decreased (P<0.05) in oocytes compared with COCs and granulosa cells. These results demonstrate specific miRNAs within bovine COCs during late oogenesis and provide some evidence that miRNAs may play a role regulating maternal mRNAs in bovine oocytes.
The objective was to determine the sire effect on the pregnancy outcome in beef cows in which stage of estrus was synchronized with progesterone based fixed-time artificial insemination (AI) protocols. Three Angus sires with more than 300 breedings were evaluated for differences in pregnancy outcome from 1868 inseminations. Angus cross beef cows (N=1868) were synchronized with Ovsynch-CIDR or CO-Synch-CIDR protocols for fixed-time AI. Cows in both groups that showed estrus on day 9 before 1500 h were designated to Selectsynch-CIDR group and were inseminated according to AM-PM rule. Results indicated that Sire 2 had lower fixed-time AI pregnancy rate compared to Sire 3 (48.1% versus 58.7%; P=0.01). Significant sirexsynchronization program and sirexlocation interactions were observed for fixed-time AI (P<0.05). Sire 2 had a lesser fixed-time AI pregnancy in both Ovsynch-CIDR and CO-Synch-CIDR groups compared to Sire 3. In two of four locations, Sire 2 had a lesser fixed-time pregnancy rate compared to Sire 3. No sire differences were observed in AI pregnancy for cows in Selectsynch-CIDR group. In conclusion, evidence in this study suggest that there are differences in sire fertility when they were used in fixed-time AI protocols, possibly due to the sire differences in sperm capacitation process. Further studies are needed to investigate association of the sire differences in fixed-time AI protocols with sire differences in the sperm capacitation process.
The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.
A study was carried out to assess the effect of sequestration of PDC-109 protein, a majority constituent of heparin binding proteins (HBP) of seminal plasma, on freezability and in vitro fertilizing ability of crossbred bull spermatozoa after cryopreservation. The study consisted of isolation and characterization of PDC-109 protein to raise anti-sera against it in rabbits. Following which, raised antibodies against PDC-109 protein was quantitated and coated in tubes used for collection of ejaculates. Semen ejaculates thus collected were cryopreserved using EYTG extender. Physico-morphological characteristics, viz. motility, viability, acrosomal integrity and HOS response as an indicator of freezability of cryopreserved spermatozoa were determined at pre freeze as well as post thaw stage. At pre freeze stage, a significant (p<0.05) improvement in viability (83.83 ± 2.18 vs 75.17 ± 2.42) and acrosome integrity (81.33 ± 2.38 vs 72.83 ± 2.39) in antibodies treated group than control was observed. Similarly, increase in HOS responsive spermatozoa was highly significant (p<0.01) than control (78.83 ± 1.69 vs 67.5 ± 1.75). At post thaw stage, significant (p<0.05) improvement in viability (69.50 ± 2.16 vs 60.33 ± 2.19) and HOS responsive spermatozoa (68.67 ± 1.62 vs 58.50 ± 1.32) and highly significant (p<0.01) increase in individual motility (56.17 ± 1.83 vs 47.00 ± 1.86) and acrosome integrity (75.17 ± 2.38 vs 61.83 ± 2.1) was observed in antibodies treated group when compared to control was observed. The results from the study revealed that sequestration of PDC-109 protein from semen samples leads to significant improvement in pre-freeze and post-thaw values of above parameters in cryopreserved spermatozoa. It is thus concluded that sequestration of PDC-109 protein from ejaculates improves freezability of crossbred bull spermatozoa.
The aim of this study was to investigate the distribution of CD2+, CD4+, CD8+ lymphocyte subpopulations and MHC class II expressing cells in the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace×Swedish Yorkshire), with an average parity number of 3.4±0.7 (mean±S.D.) were used. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus (day 17, n=3), prooestrus (day 19, n=3), oestrus (day 1, n=3), early dioestrus (day 4, n=3) and dioestrus (days 11–12, n=3), were stored in a freezer at −70 °C until analysed by immunohistochemistry with an avidin-biotin peroxidase method using monoclonal antibodies to lymphocyte subpopulations and MHC class II molecules. The surface and glandular epithelium as well as connective tissue layers in subepithelial and glandular areas were examined by light microscopy.
An excessive lipid content in embryo cells is a consequence of embryo culture in the presence of serum which is suggested to be responsible for their high susceptibility to cryopreservation. The objective of the present study was to examine the effects of supplementing serum-containing culture media with trans-10 cis-12 conjugated linoleic acid (10t,12c CLA) on embryo lipid accumulation and its subsequent cryopreservation. Abattoir-derived oocytes were matured and fertilized in vitro (IVF=day 0). On day 1, presumptive zygotes (n=3390) were randomly placed in: (I) (MS), granulosa cell monolayer cultured with M199 and 10% serum; (II) (SCLA), granulosa cell monolayer cultured with M199, 10% serum and 100 microM 10t,12c CLA and (III) (SOF), modified synthetic oviduct fluid, where embryo culture proceeded for 8 days. Cleavage rates or D7/D8 embryo quality did not vary among treatments. D7/D8 embryo production rate was significantly (P<0.001) lower in SOF (17.9+/-1.6%) than in groups MS (29.8+/-2.5%) and SCLA (27.8+/-2.0%). After cytoplasmic lipid droplets observation under Nomarski microscopy, classified embryos were the leanest when cultured in SOF, intermediate in SCLA and the fattest in MS (P<0.02). Post-thawing intact blastocyst rates where significantly higher in the SCLA group (84.7+/-4.1%) than in SOCS (50.3+/-4.8%, P=0.0007) or SOF (65.3+/-6.9%, P=0.03) groups. Post-thawing re-expanding rates were significantly lower when embryos were cultured in MS (34.7+/-3.7%) than in SCLA (63.7+/-5.3%, P=0.0006) or SOF (49.0+/-4.6%, P=0.04). Moreover, re-expanding rates were lower (P=0.05) in SOF than in SCLA cultured embryos. These results clearly show that addition of CLA to serum-containing media reduced lipid accumulation during in vitro culture and significantly improved cryopreservation survival.
The aims of this study were to determine: (1) if short-term treatment of Bos indicus heifers with progesterone (P4) while implanted with a s.c. norgestomet implant for 17 days would influence the time interval to oestrus and increase fertility of the synchronised oestrus, and (2) whether the response to treatment with P4 would differ between heifers treated with a norgestomet implant for 17 vs. 11 days when short-term treatment with P4 is applied 3 days prior to implant removal. B. indicus heifers at two separate sites (A and B) were allocated to three groups at each site. Heifers in two groups (NG and NGP4 groups) were given a single s.c. norgestomet implant on the first day of treatment (day 0) while heifers in a third group (NGP4PG group) were implanted on day 6. A single P4 releasing Controlled Internal Drug Release device (CIDR) was inserted on day 14 in heifers in the NGP4 and NGP4PG groups and was removed 23.5 +/- 0.07 h later (day 15). Heifers in the NGP4PG group were administered an analogue of prostaglandin F2 alpha (PGF2 alpha) at the time of CIDR removal to regress corpora lutea. Implants were removed from all heifers on the same day (day 17) and a 400 IU of equine chorionic gonadotrophin (ECG) was administered s.c. Animals were artificially inseminated 11.1 +/- 0.17 h after detection of oestrus, using frozen semen from one bull at site A and one of five bulls at site B. Inseminations were carried out by one of two technicians. Treatment with P4 delayed oestrus and reduced the synchrony of oestrus at site A (hours to oestrus +/- SD: NG group, 39.0 +/- 13.7; NGP4 group, 66.3 +/- 24.4; NGP4PG group, 58.9 +/- 20.5 h; P < 0.05) but not at site B (41.4 +/- 15.2, 42.5 +/- 10.1, 45.4 +/- 10.3 h; P > 0.05). Pregnancy rates 6 weeks after insemination were found to be significantly associated with bull (P < 0.001), treatment group (P = 0.013) and insemination technician (P = 0.033). Pregnancy rates were greater in the heifers in the NGP4 group than heifers in the NG group [50.3% (78/155) vs. 36.4% (60/165); odds ratio = 1.83, 95% CI = 1.14 to 2.96] and similar between heifers in the NGP4 and NGP4PG groups [50.3% (78/155) vs. 51.1% (63/117); odds ratio = 1.06, 95% CI = 0.67 to 1.69]. It was concluded that acute treatment with P4 can improve pregnancy rates in B. indicus heifers treated for 17 days with norgestomet implants. Reducing the duration of norgestomet treatment to 11 days and administration of PGF2 alpha at the time of ending treatment with a CIDR device resulted in no differences in fertility, mean intervals to oestrus or synchrony of oestrus.
This study determined the relationship between two measures of field fertility of 11 high-use Australian artificial insemination (AI) dairy bulls and thirty standard laboratory assessments of spermatozoal post-thaw viability. The two measures of field fertility used, conception rates (cCR) and non-return rates (cNRR), were both corrected for all major non-bull variables. Sperm viability assessments were conducted on semen collected within the same season as that used to derive the field fertility estimates. These assessments measured sperm concentration, motility, morphology and membrane integrity at thawing, after 2h incubation and after the swim-up sperm selection procedure. Derivations of these measures and in vitro embryo fertilizing and developmental capacity were also determined. The Genstat Statistical Package [Genstat 5 Release 4.2 Reference Manual, VSN International, Oxford, 2000] was used to conduct an analysis of variance on the viability parameters across semen straws and bulls, and to calculate the strength of correlation between each semen parameter, cNRR and cCR in a correlation matrix. Step forward multiple regression identified the combination of semen parameters that were most highly correlated with cCR and with cNRR. The sperm parameters identified as being most predictive of cCR were the percentage of morphologically normal sperm immediately post-thaw (zeroNorm), the number of morphologically normal sperm after the swim-up procedure (nSuNorm), and the rate of zygote cleavage in vitro (Clv); the predictive equation formed by these parameters accounted for 70% of variance. The predictive equation produced for cNRR contained the variables zeroNorm, the proportion of membrane intact sperm after 2h incubation at 37 degrees C (twoMem) and Clv and accounted for 76.5% of the variation. ZeroNorm was found to be consistent across straws and semen batches within-bull and the sperm parameter with the strongest individual predictive capacity for both cCR (P=0.1) and cNRR (P=0.001). Post-thaw sperm parameters can be used to predict field fertility of Australian dairy sires; the calculated predictive equations are particularly useful for identifying and monitoring bulls of very high and very low potential fertility within a group.
The aim of present work was to analyze the body reserves and ovarian features of lactating primiparous rabbit does under extensive reproductive management (artificial insemination (AI) at 25 days post-partum (dpp)) compared with the common insemination rhythm at 11 dpp. A total of 48 primiparous Californian×New Zealand White rabbit does suckling 8 kits were used to assess liveweight, estimated body composition, serum metabolic and endocrine parameters (oestradiol and progesterone concentrations) and ovarian features like follicle population and atresia rate, and oocyte maturation. Rabbit does were randomly allocated in two experimental groups: (a) lactating does euthanized at early post-partum period (11 dpp) according to a semi-intensive rhythm (n=24), and (b) lactating does euthanized at later post-partum period (25 dpp) according to a more extensive rhythm (n=24). Liveweight, body energy content, lipid depots and serum non-esterified fatty acids (NEFA) concentrations decreased from parturition to post-partum period (P<0.05). In addition, serum protein and glucose concentrations increased in the post-partum period (P<0.05). Similar oestradiol and progesterone levels were found in rhythms as well as similar follicle population and nuclear and cytoplasmic maturation rates measured as metaphase II and cortical granule migration, respectively in both post-partum times. However, the number of preovulatory follicles on the ovarian surface was lower (P<0.05) and the atresia rate tended to be higher with a lower percentage of healthy follicles (P<0.1) in ovaries from females of extensive group. In conclusion, the body reserves, serum metabolic parameters and oocyte quality of primiparous non-weaned rabbits does at the late post-partum time (25 days) were not improved. Thus this reproductive management did not present any advantages compared to earlier post-partum (11 days) reproductive rhythm.
To better understand the role of estradiol-17beta in fetal ovarian development, presence and localization of cytochrome P450 aromatase (P450arom) and estrogen receptors alpha (ERalpha) and beta (ERbeta) proteins were characterized in fetal ovaries of cattle using immunohistochemistry. Fetal cattle ovaries were collected from an abattoir and sorted into fetal age groups (days 110, 130, 150, 170, 190, 210, 230, 250+) based on crown-rump length. In addition to immunohistochemistry, morphological analysis of ovarian and follicular formation was made. Ovaries appeared lobular at day 110, but by the end of gestation (day 250+) ovaries were oval-shaped similar to those found in adult animals. Ovarian structures within different lobes appeared to be at different developmental stages. At day 110, oocytes and pre-granulosa cells were observed in ovigerous cords that were still open to the surface epithelium. Most ovigerous cords appeared to be closed to the surface epithelium on day 130, all closed by day 150 and were no longer present at day 210. Ovarian follicles were classified as follows: Type 1(primordial): single layer of flattened granulosa cells, Type 1a (transitory): single layer of mixed flattened and cuboidal granulosa cells, Type 2 (primary): at least one but less than two layers of cuboidal granulosa cells, Type 3 (small preantral): two to three layers of granulosa cells, Type 4 (large preantral): four to six layers of granulosa, and the theca layer is forming around the follicle, Type 5 (antral): contain greater than six layers of granulosa cells, several layers of theca cells and the antrum has formed. Type 1 follicles were observed in day 110 ovaries. Follicle Types 1a and 2 were first observed on day 130. Type 3 follicles were first observed on day 150 and Types 4 and 5 were first observed on day 170. P450arom protein was localized in granulosa cells of follicle Types 2-5 and cells of rete tubules throughout the experimental period. There was punctate expression within stroma and rete masses. There was ERalpha protein localization in pre-granulosa cells and germ cells of ovigerous cords and all surface epithelial cells. There was also localization in granulosa cells and oocytes of all follicle types and cells of rete tubules. There was punctate ERalpha protein expression in stroma and rete masses. ERbeta protein was localized in pre-granulosa cells and germ cells of ovigerous cords. Expression was also localized to granulosa cells of all follicle types and cells of rete tubules. ERbeta protein was punctate in oocytes of follicles, surface epithelial cells, stroma and rete masses. Thus, the fetal ovary of cattle has the steroidogenic enzyme (P450arom) to convert androgens to estradiol-17beta, and estrogen receptors alpha and beta to facilitate an estrogen response within the fetal ovary.
The aim of this study was to investigate the distribution of leukocytes and the morphological changes of the sow endometrium throughout the oestrous cycle. Fifteen crossbred multiparous sows (Swedish Landrace × Swedish Yorkshire), with an average parity number of 3.4±0.7 (mean±S.D.) were used. Blood samples were collected from the jugular vein 1 h before slaughter for analyses of oestradiol-17β and progesterone levels. Uterine samples from the mesometrial side of both horns, taken immediately after slaughter at late dioestrus, prooestrus, oestrus, early dioestrus and dioestrus, were fixed, embedded in plastic resin and stained with toluidine blue. The surface and glandular epithelium as well as subepithelial and glandular connective tissue layers were examined by light microscopy (LM).
Vascular endothelial growth factor (VEGF) is a potent stimulator of endothelial cell proliferation and neo-vasculogenesis. In the ovary, VEGF mRNA is localised in the follicle, and it is associated with follicular growth and dominance. Alternative splicing of VEGF mRNA produces eight mature forms of mRNA for equal number of VEGF isoforms. In the present study, the VEGF isoforms in granulosa and theca cells of large (4-6mm) and preovulatory (>6mm) sheep follicles were studied during the process of atresia. Follicles were classified as healthy, early atretic and atretic, and the granulosa and theca cells were isolated. The mRNA for three of these isoforms was found in both theca and granulosa cells, and was quantified by image analysis after RT-PCR using primers that amplified VEGF120, VEGF164, VEGF188 and VEGF205 isoforms. The mRNA for these three isoforms was found in both theca and granulosa cells of healthy and atretic follicles. Atresia was accompanied with a reduction in mRNA for VEGF164 and VEGF120 in granulosa and theca cells (P<0.05). Amounts of both isoforms were reduced with the extent of atresia in the granulosa cells, whilst in the theca cells this reduction was only evident in advanced atretic follicles. Furthermore, after the onset of atresia, VEGF205 was not detectable in the granulosa cells. Follicle size did not affect the amount of VEGF mRNA. Hence, the onset of atresia in follicles of sheep is coupled with a reduction in VEGF mRNA. The decrease in VEGF observed with atresia in follicles of sheep was greater in granulosa than in theca cells.
To control postpartum anestrus and reduce calving to conception interval, 167 crossbred non-pregnant cows that were 90-130 days postpartum were allotted randomly to one of the following treatments: PH (n=59), intra-vaginal sponge with 250 mg of medroxyprogesterone acetate (MAP) for 7 days plus 50mg of MAP and 5mg 17-beta estradiol (17beta-E) in the first day of treatment (day -8), 500 UI eCG (day -3) and 1.5mg 17beta-E in 24h after sponge removal (day 0); CR (n=57), temporary calf removal for 120 h; CG (n=51), control group without treatment. Estrus rate differed among treatments (P<0.01) being greater in PH (78.2%), followed by CR (52.0%) and CG (22.9%). A greater proportion of cows in the PH (80.0%) and CR (54%) groups had ovulations when compared to CG (35.4%). Intervals to first estrus were 13.5+/-6.3 days, 26.1+/-6.4 days and 52.5+/-7.5 days for the PH, CR and CG groups, respectively. First insemination conception was similar in the three groups. Postpartum intervals to first breeding (PFS) and to conception (PCI) were longer in CG than PH and CR groups (P<0.05; P<0.01). The PH and CR groups had a similar PFS but PCI was different (P<0.02). Accumulated pregnancy rate at 30 and 60 but not at 90 days were different (30 days: P<0.09; P<0.01; P<0.09; 60 days: P<0.06; P<0.01; P<0.03) among treatments. After 90 days post-treatment, 9%, 18% and 33% of cows from the PH, CR and CG groups had not conceived. Similarly, 5.4%, 6.0% and 12.5% of cows from the PH, CR and CG groups, respectively, were culled from the herd because of lack of pregnancy after 180 days post treatment. In the group of cows evaluated by ultrasonography, only those cows having larger ovaries and dominant follicles had ovulations. It was concluded that the hormonal treatment was more efficient in inducing a fertile estrus and reducing calving to conception interval followed by the calf removal for 120 h. Each method can be considered as an important tool to reduce the postpartum anestrous period in dual purpose herds when AI is conduct in the tropics.
Vascular endothelial growth factor (VEGF) isoforms (VEGF 120 and VEGF 164) secreted by granulosa cells are involved in thecal angiogenesis during follicular development in the bovine ovary. The follicular fluid in the developing follicle includes a slight amount of the progesterone. However, the progesterone (P4) effects on VEGF120 and VEGF164 isoforms have not been well characterized in the bovine granulosa cells. We investigated the effects of progesterone on the gene expression of hypoxia-inducible factor 1alpha (HIF-1alpha, transcription factor), VEGF120, VEGF164 and Flk-1, its receptors, in cultured bovine granulosa cells. Messenger RNA expression for HIF-1alpha, VEGF120, VEGF164 and Flk-1 was quantified using real-time PCR methods. The levels of VEGF120, HIF-1alpha and Flk-1 mRNAs were increased significantly by P4 at a concentration of 10 ng/ml. In contrast, the expression of VEGF 164 gene is inhibited by P4. The level of VEGF120 and Flk-1 mRNAs in the granulosa cells treated with 10 ng/ml progesterone plus 1 ng/ml estradiol significantly decreased compared with progesterone alone. In contrast, the addition of 1 ng/ml estradiol to the culture medium increased the expression of VEGF164 gene. In conclusion, our data demonstrated that progesterone might stimulate the expression of the VEGF120 via HIF-1alpha, transcription factor, in bovine granulosa cells. These results suggest the hormone-dependent expression pattern of VEGF isoforms during follicular development. Thus, our study suggested the expression of VEGF isoforms in granulosa cells might be controlled by a different pathway during follicle development in cow.
Twenty-one Pelibuey ewes were used from December 21, 1996 to December 21, 1998. Fourteen of them had never been exposed to artificial photoperiod, and they were maintained on natural photoperiod until March 21, 1997, when they were assigned to natural photoperiod (control group, n=8) or to inverse photoperiod (n=6). The other seven animals had been kept on a long photoperiod (16L:8D) from October 21, 1996 to December 21, 1996, when they entered the present study and were subjected to a gradual decrease in photoperiod, so that they reached an equinox photoperiod (12L:12 D) on March 21, 1997. At that time, they were assigned to natural photoperiod (n=3) or to inverse photoperiod (n=4). Blood samples for progesterone determination were taken twice a week from all the animals. During the second year of the study, prolactin was measured in the samples from five animals in inverse photoperiod and from five control ewes. Hourly samples were obtained to determine the 24-h melatonin profile of five animals from each group on September 21, 1997, December 21, 1997, March 21, 1997, and June 21, 1997. Exposure to inverse photoperiod resulted in a gradual shift on the annual reproductive cycle, so that the second ovulatory season was advanced by 5 months in the ewes kept on inverse photoperiod as compared to the control ewes (P<0.05). There were wide variations in the dates for the onset and the end of the ovulatory season within the inverse photoperiod groups, and three animals in this groups maintained ovulatory activity for at least 18 consecutive months. The duration of melatonin secretion was directly related to the length of the dark period (P<0.05), and this response was not affected by the calendar date. Prolactin concentrations were directly related to daylength, however, they were also affected by calendar date, being lower in the inverse group as compared to the corresponding time of the annual photoperiodic cycle of ewes on natural photoperiod. It is concluded that reproductive activity, melatonin secretion and prolactin secretion of Pelibuey ewes respond to the small variations in photoperiod that are present at 19 degrees 13'N, and that under natural conditions, photoperiod appears to be the main regulator of ovarian activity at this latitude. However, other factors such as temperature or humidity may act as modulators, and their relative importance could increase at more equatorial latitudes.
Twenty-one of the world's prolific sheep breeds and strains were tested for the presence of the FecB mutation of BMPR1B and the FecX(I) mutation of BMP15. The breeds studied were Romanov (2 strains), Finn (2 strains), East Friesian, Teeswater, Blueface Leicester, Hu, Han, D'Man, Chios, Mountain Sheep (three breeds), German Whiteheaded Mutton, Lleyn, Loa, Galician, Barbados Blackbelly (pure and crossbred) and St. Croix. The FecB mutation was found in two breeds, Hu and Han from China, but not in any of the other breeds. The 12 Hu sheep sampled were all homozygous carriers of FecB (FecB(B)/FecB(B)) whereas the sample of 12 Han sheep included all three genotypes (FecB(B)/FecB(B), FecB(B)/FecB+, FecB+/FecB+) at frequencies of 0.33, 0.58 and 0.08, respectively. There was no evidence of FecX(I) in any of the breeds sampled.
Group housing of sows during the mating and gestation period has become the overall common management practice in Sweden. Loose housing is probably less stressful for the animals because it allows them more opportunities to behave naturally, but mixing unfamiliar sows does create a stressful situation due to aggressive interactions, which can lead to food deprivation. The objective of the present study was to investigate and compare the effects of stress in form of food deprivation and ACTH administration at days 13 and 14 of pregnancy (day 1, first day of standing oestrus) in sows. The hormonal secretion of the sows and foetal survival by day 30 of pregnancy was, therefore, studied in 17 crossbred multiparous sows. The sows were randomly allocated into three different groups: one control (C-) group; one food deprived (FD-) group, which was deprived of food from the morning of day 13 of pregnancy until the evening meal on day 14; and a third group (A-), which was given intravenous injections of synthetic ACTH (Synachten Depot), at a dose of 0.01 mg/kg body weight every sixth hour from 6 a.m. on day 13 until 6 a.m. on day 15 of pregnancy. All sows were slaughtered at 30 +/- 2 day of pregnancy and the genital tracts recovered. Total number of corpora lutea (CL), total number of viable or nonviable embryos and foetal survival rates were determined. Samples from the peripheral blood circulation were collected four times a day from day 12 until slaughter, except during days 13-15 when blood was collected every second hour. The blood samples were analysed for cortisol, progesterone, oestrone, prostaglandin F(2alpha)-metabolite, oestrone-sulphate, insulin, free fatty acids and triglycerides. FD-sows had increased levels of cortisol, free fatty acids and progesterone, as well as a lowered level of insulin in the peripheral blood plasma, while A-group sows had increased levels of both cortisol and insulin compared with the C-group. Treatment with ACTH seemed to cause a 2-day delay in the increase of oestrone, from day 19, as seen in the FD- and C-group, to day 21 of pregnancy. At the time of slaughter, there were no significant differences among groups in terms of total number of foetuses and foetal survival rate. The results of the present study suggest a capacity of the sow to compensate for the influence of induced moderate stress at the time of pregnancy when maternal recognition occurs.
A simple, sensitive and direct enzymeimmunoassay (EIA) procedure on microtitre plates using the second antibody coating technique was standardized and validated for the determination of 13,14-dihydro-15-keto PGF2alpha (PGFM) in unextracted buffalo plasma. The assay was carried out directly in 20 microl of buffalo plasma. PGFM standards prepared in charcoal stripped hormone-free plasma were used. The sensitivity of the assay was 0.4 pg/well, which corresponded to 20 pg/ml plasma. Plasma volumes for the assay ranging from 10 to 50 microl did not influence the PGFM standard curve; however, a slight drop in the OD450 was observed with higher plasma volumes. Biological validation of the assay was carried out in buffalo plasma samples obtained during physiological states of cyclicity, peri-estrus, post-insemination, reproductive tract infection and persistent corpus luteum conditions. A pulsatile pattern of plasma PGFM release was observed prior to estrus when PGFM was determined in blood samples collected at hourly intervals of time. The PGFM pulsatility was not observed when blood sampling frequency of either 4 or 12 h was considered. The PGFM levels stayed high in peripheral circulation of buffaloes with reproductive tract infections and remained low throughout the sampling period in buffaloes having persistent corpus luteum. After an initial increase post-insemination, the plasma PGFM levels showed minor fluctuations. The assay was found to be sufficiently reliable and specific for estimation of PGFM levels in buffaloes. The standardization and validation of PGFM assay in buffalo opens the prospects of using PGFM levels as an indicator for reproductive health status monitoring in this species.
Plasma concentrations of progesterone, oestradiol-17beta, oestrone, oestrone sulphate and PGFM have been measured daily during the first peri-partum period of 45 Hereford x Friesian heifers bred at 11 months of age. Anatomical measurements of dam and calf were also recorded. Twelve of the calvings were scored easy, 33 difficult. Each of five models (fitted by linear logistic regression) relating difficulty of calving to the hormonal and anatomical measurements, predicts with at least 94% accuracy the calving score (easy or difficult) among the calvings. The models predict that increases of progesterone concentration on the day before calving, of oestrone sulphate concentration on the day after calving and of heifer heart girth decrease the odds of difficult calving, whereas increases of heifer body length and of calf head circumference increase the odds of difficult calving.
The objective of the present study was to validate a simple, sensitive and direct enzymeimmunoassay (EIA) procedure for 13,14-dihydro-15-keto-PGF(2alpha) (PGFM) for use in buffaloes with postpartum reproductive disorders and determine the practicalities of using plasma concentrations of 13,14-dihydro-15-keto-PGF(2alpha) for monitoring their reproductive health. The EIA was used for determination of the circulating levels of PGFM associated with the retention of fetal membranes, postpartum endometritis and variable postpartum intervals. The concentrations of PGFM with retention of fetal membranes in the periparturient period were lower as compared to buffaloes that had uneventful parturitions. Concentrations of PGFM associated with postpartum endometritis were elevated as compared to those in buffaloes free of reproductive tract infections. Buffaloes having higher plasma concentrations of PGFM in early postpartum period had shorter postpartum intervals, indicating the association between PGFM concentrations postpartum and uterine involution as well as the resumption of estrous cycle in this species. The study presents the possibility of using circulating PGFM concentrations for monitoring the postpartum reproductive health of buffaloes.
Staining with Hoechst 33342 followed by ultraviolet irradiation is frequently used to aid or confirm the enucleation of recipient oocytes in porcine somatic cell nuclear transfer programs. However, the procedure has a clearly deleterious effect on the developmental ability of oocytes. This study evaluated the effectiveness of a longer-wavelength fluorochrome (SYBR-14) for visualizing maternal chromosomes in in vitro-matured porcine oocytes and the effects of this dye in combination with fluorescence excitation on the subsequent in vitro fertilization and embryo development of the oocytes. In the first experiment, the oocytes were exposed to different concentrations (1, 3, 5 and 7 μg/mL) of SYBR-14 at different incubation times (5, 10 and 30 min) in a 4 × 3 factorial design. The optimal condition for proper metaphase-II plate and first polar body visualization was a 10-min incubation with 5 μg/mL of SYBR-14. In the second experiment, the degeneration rate of the oocytes 18 h after exposure to SYBR-14 (5 μg/mL for 10 min) and fluorescence excitation for 5 or 30s was significantly higher (p<0.002) than that obtained for non-exposed oocytes. The fertilization parameters were not influenced by the treatments. The cleavage and blastocyst rates during culture were lower (p<0.001) for the oocytes exposed to SYBR-14 and fluorescence than for those in the non-exposed group. These results indicate that the exposure of mature oocytes to SYBR-14 and fluorescence for periods as short as 5s increased the rate of oocyte degeneration and limited their subsequent developmental competence.
Continuous GnRH agonist treatment of cows results in downregulation of GnRH responsiveness and a state of induced anoestrus. Inducing anoestrus in a precisely controlled manner could have several potential applications in dairy herd management. However, relatively little is known regarding the processes involved in restoring reproductive normality following an induced anoestrus. This study describes an experiment that was conducted to examine patterns of recovery of LH release and follicle growth in non-lactating Holstein cows immediately following cessation of treatment for 7, 14 or 21 days with a deslorelin implant. Oestrus cycles were synchronized at 7 days intervals and a deslorelin implant inserted in every cow 13 days after detected oestrus so that a group had implants for either 21 days (n = 9), 14 days (n = 10) or 7 days (n = 9). On the day of implant removal every ovarian follicle greater than 4 mm in diameter was ablated using ultrasound guided vacuum needle aspiration in an attempt to standardize follicle sizes. Daily ovarian ultrasound examinations were performed on each cow until 35 days after implant removal and again at 45, 59 and 74 days. A subgroup of four cows randomly selected from each treatment group had frequent serial blood samples collected over 8 h at 4 and 10 days after implant removal for LH profiling.
The objective of the study was to compare the systemic antibiotic treatment of clinical endometritis in dairy cows with ceftiofur with a treatment protocol consisting of two doses of prostaglandin F(2alpha) analogue cloprostenol in a 14-d interval. On 2 commercial dairy farms, housing a total of 1900 Holstein cows, all cows that calved between June 2008 and January 2009 were examined 21-27d in milk (DIM) by vaginoscopy. Cows with clinical signs of endometritis, i.e. vaginal discharge containing flecks of pus, mucopurulent material or purulent mucus, were randomly allocated to one of two treatment groups. Cows in group CEF (n=141) received 1 mg/kg BW of ceftiofur (i.m.) on 3 consecutive days. Cows in group CLP (n=140) received 0.5 mg of cloprostenol (i.m.) at the day of enrolment and 14d later. All cows were re-examined by vaginoscopy 42-48 DIM. Proportion of cows cured, i.e. cows with clear, translucent or no mucus, 42-48 DIM (74.2 and 80.2% in groups CEF and CLP, respectively) was not affected by treatment group (P=0.09). The voluntary waiting period was set at 40 DIM. Artificial insemination (AI) submission rate, days to first service, first service conception rate, days open and proportion of cows pregnant did not differ between the groups. In conclusion, the systemic treatment with 1.0 mg/kg BW of ceftiofur on 3 consecutive days in cows with signs of clinical endometritis 21-27 DIM was equivalent to an intervention protocol consisting of two doses of cloprostenol in a 14-d interval.
Gonadotropin releasing-hormone analogue (buserelin) challenges were carried out every 8 weeks from 4 to 14 months of age on thoroughbred colts born in the spring (n = 6) or autumn (n = 5) to define the onset of puberty. In all colts, luteinizing hormone (LH) secretion followed a seasonal pattern, with high baseline and maximal concentrations in the spring and summer and low concentrations in the winter. Testosterone concentrations were undetectable before spring and, thus, autumn-born colts were younger than spring-born colts when a testosterone response to buserelin was first observed. Mean weights of the autumn-born colts were 300 kg (282-327 kg) at the time of the first detectable testosterone response in the following spring (October). Spring-born colts had reached this weight in the winter (May and June, before day length had increased) but did not exhibit a significant testosterone response until the spring at a mean weight of 352 kg (327-403 kg). It is proposed that colts must achieve a threshold body weight concurrently with stimulatory photoperiod for onset of puberty to occur.
Following insemination, reproductive failure in cattle is largely manifested as embryo mortality and is a major source of financial loss to livestock producers. Ongoing studies at this laboratory into factors affecting embryo mortality have facilitated the collection of new data on the extent and timing of embryo and foetal mortality in cattle. Oestrus was synchronised in 158 beef cross heifers and following artificial insemination, embryo and foetal survival rates were determined on days 14 and 30 after insemination and subsequently at calving. Embryo survival rates measured on days 14, 30 and at full term were similar at 68%, 76% and 71.8%, respectively (P0.05). Based on morphological examination, all the 14-day-old embryos recovered were assessed as grade 1. These results provide new information indicating that most embryo losses in heifers have occurred before day 14 after insemination.
The goal of the present study was to characterize the semen quality of dogs naturally infected with Leishmaniachagasi, and treated with Allopurinol and Amphotericin B. Eight naturally infected and eight non-infected dogs were selected. Following semen collection, progressive motility, vigor, concentration and sperm morphology were evaluated. The seminal patterns in the treated animals were evaluated at the beginning (d0) and at days 30 (d30), 60 (d60) and 150 (d150) of treatment. The progressive motility at d0 (35.7+/-22.3%) was less than that of the control group (77.8+/-7.1%) (P<0.05). The vigor was similar to the control group throughout the treatment (P>0.05). The number of sperm/mL, sperm/ejaculate and sperm/kg of body weight was similar among groups (P>0.05). The percentages of normal spermatozoa of infected and treated animals were similar throughout the treatment and to the control group (69.1+/-8.7%) at d60 (37.5+/-11.2%) and d150 (48.3+/-10.8%) (P>0.05), but smaller at d0 (22.7+/-10.5%) and d30 (28.8+/-15.9%) (P<0.05). A greater percentage of acrosome damage was observed in the control group (3.1+/-2.3%) compared to the d60 (0.1+/-0.2%) (P<0.05). The infected dogs had a greater percentage of principal piece defects at d60 (37.0+/-6.3%) than the control group (16.8+/-7.3%) (P<0.05); and greater percentages of detached normal heads at d0 (28.7+/-19.7%) and d30 (18.5+/-18.5%) than the control group (0.4+/-0.5%) (P<0.05). This reduction in semen quality of the infected animals is suggestive of an epididymal dysfunction. Due to this poor semen quality, caution is recommended when using infected male dogs for reproductive purposes.
Different mutations in the Bone Morphogenetic Protein 15 (BMP15) and the Growth Differentiation Factor 9 (GDF9) genes cause increased ovulation rate and infertility in a dosage-sensitive manner in sheep. They cause increased ovulation rate and twin and triplet births in heterozygotes, and complete primary ovarian failure in homozygotes resulting in total infertility. We are here presenting a novel mutation in the second exon of the ovine BMP15 gene, found in the Spanish breed Rasa Aragonesa. It consists of a 17 bp deletion resulting in displacement of the open reading frame and premature stop codons. As a consequence, nearly 85% of the sequence of the wild type aminoacidic chain in the second exon of the BMP15 pro-protein is modified or suppressed as only the first 45 amino acids are conserved of the 245 original. The mature peptide is lost. The ewes heterozygous for this deletion present very high prolificacy (2.66 lambs/birth) when compared to a mean flock prolificacy of 1.36 lambs. The deletion causes a complete lack of functionality of the second exon of BMP15, comparable to the effect of premature stop codons in other mutations. Therefore, homozygous females for the deletion are expected to present primary ovarian failure. DNA sequence analysis of the GDF9 coding regions detected only a synonymous Single Nucleotide Polymorphism (SNP), apparently not linked to changes in prolificacy.
Two experiments were designed to determine if the suppressive effect of estradiol treatment on ovarian follicles in progestogen-implanted heifers is mediated directly at the ovary or systemically, at a higher level. The purpose of Experiment 1 was to determine a minimal effective dose of estradiol-17β (E-17β) that would induce follicle regression in progestogen-implanted heifers. Beef heifers were implanted with progestogen on Day 2 (Day 0=ovulation) and were assigned randomly to five groups: control (sesame seed oil, n=9); 0.1 mg of E-17β (n=8); 0.5 mg of E-17β (n=8); 1 mg of E-17β (n=8); or 5 mg of E-17β (n=8) by intramuscular (im) injection on Day 3. Treatment with 5 and 1 mg of E-17β resulted in smaller (P
Ultrastructure of sterlet Acipenser ruthenus L. 1758 sperm was examined by scanning and transmission electron microscopy, which allowed us to use various methods for visualizations of different parts of sterlet spermatozoa. Sperm cells possess a head with a distinct acrosome, a midpiece and a single flagellum surrounded by the flagellar plasma membrane. The average length of the head including the acrosome and the midpiece was estimated as 5.14+/-0.42 microm. Nine to 10 posterolateral projections were derived from the acrosome. Three inter-twining endonuclear canals bounded by membranes traversed the nucleus in its whole length from the acrosome to the implantation fossa. Acrosin was located in all the three parts (acrosome, endonuclear canals and implantation fossa). The proximal and distal centrioles located in the midpiece compacted of nine peripheral triplets of microtubules. One cut of the midpiece contained from two to six mitochondria with area of 215+/-85 nm(2) in average. The flagellum was 42.47+/-1.89 microm in length with typical eukaryotic organization of one central pair and nine peripheral pairs of microtubules. It passed through a cytoplasmic channel in the midpiece, which was formed by an invagination at the plasmalemma. The flagellum gradually developed two lateral extensions of its plasma membrane, so-called "fins". Detected morphological variation can be described by four principal component axes corresponding to groups of individual morphometric characters defined on the sperm structures. Correlations among the characters indicate that the sperms are variable in their shape rather than size. Significant variation among examined fish individuals was found only in flagellum and nucleus length. Comparison between the present and previous studies of morphology of sturgeon spermatozoa confirmed large inter- and/or intra-specific differences that could be of substantial taxonomic value.
The aim of this research was to study the individual variation with regard to the morphometry of the testes evaluated by ultrasonography and semen characteristics and to verify the existence of relationship between these variables in collared peccaries. In addition, the testes of the animals were evaluated by histology in order to determine the proportion occupied by the seminiferous tubules. A total of 52 ejaculates were obtained from ten adult specimens that had been restrained by anesthesia. The testicular measurements (length, height, and width) were performed by ultrasonography, and the testicular volume was calculated according to Lambert's formula. The scrotal circumference was measured by encircling the thickest portion of the testicle with a graduated nylon tape. The semen was collected by electroejaculation. Testicular fragments were analyzed through classic histology for the determination of the area occupied by the seminiferous tubules. The results show a great amount of individual variation with regard to testicular morphometry and semen characteristics. No significant correlations were obtained between testicular measurements and semen characteristics. The histometric analysis revealed that 67.8% of the testes are occupied by seminiferous tubules. Results show that the measurement of testicular dimensions does not serve as an indicator of the quality of semen obtained by electroejaculation in collared peccaries, as there is no correlation between testicular morphometry and semen characteristics in this species that presents large variations among individuals.
Three diets were compared for the feeding of captive common sole broodstock (Solea solea) kept under ambient photoperiod and temperature conditions. A group of 70 adults were caught in the wild and the 38 males and 32 females distributed at random in six tanks. All the fish were acclimated to the same semi-moist diet (M) in the pre-experimental period from December to February. Three dietary treatments were offered in the experimental period from March to May with two replicates (tanks) per treatment. The treatments were M alone, M supplemented with fresh mussels (Mytilus edulis) (M+M), and M supplemented with live polychaetes (Perinereis cultrifera) (M+P). Spawning occurred during April and May when water temperature was 17 and 18 degrees C, respectively, and salinity around 34-35ppt. Average daily dry matter intake expressed as a proportion of body weight was M 0.65+/-0.34%, M+M 0.43+/-0.18%, and M+P 0.56+/-0.27%, and differed significantly (P<0.05) between treatments. The average daily dry matter intake within a tank ranged from 0.31+/-0.04% in February to 0.98+/-0.26% in May (P<0.05), apparently due to changes in the photo-thermal regime. Diet significantly affected the number of days when spawning occurred, the number of days when hatched eggs were produced, and the proportion of fertilized eggs (P<0.05); and affected the number of days on which viable eggs were produced during April (NS). In all cases, the results were lowest for M+M, while those for the other two treatments did not differ significantly. Differences in hatching rate were not significant in April. During May, no spawning occurred in fish given the M+M treatment, and the differences between the other two treatments were not significant. Values for all variates tended to be higher for M+P than M+M in April and lower for M+P and M in May. These results suggest that supplementing the semi-moist diet with mussels depressed feed intake and, consequently, reproductive performance; the semi-moist diet alone and semi-moist diet supplemented with polychaetes allowed satisfactory food intake and reproductive performance in broodstock sole.
The intrinsic yield of spermatogenesis and the supporting indexes of the Sertoli cells are the best indicators for the spermatic production capacity in a species. The aim of the present study was to quantify the intrinsic yield of the spermatogenetic process, as well as the Sertoli cell index and spermatic reserves. Testicular fragments of five adult African lions was fixed in 4% glutaric aldehyde, dehydrated at increasing alcohol concentrations, included into hydroxyethyl methacrylate, and were cut into 4 microm thickness. In the seminiferous epithelium of the African lions, 10.3 primary spermatocytes at pre-leptotene phase are produced by the type-A spermatogonia. During meiotic divisions, only 2.7 spermatids were produced from the primary spermatocytes. The general spermatogenesis production in the African lions was approximately 22.1 cells, and each Sertoli cell was able to sustain and maintain approximately 14.9 cells of the germinative line, from which 7.9 are round spermatids. A total of 103x10(6) spermatozoa are produced by each testis gram at each cycle of the seminiferous epithelium. The spermatic reserve of lion is below the amplitude observed in mammals.
The spotted paca is the second largest rodent in Brazil, where it is of great economic interest in impoverished regions in view of its prominence as a low-cost source of protein. Little is known about the morphology of the accessory genital glands of this species. Thus, we studied the position and morphology of the genitals in ten adult male spotted pacas. The animals were divided into two groups, five animals were used for fixing of samples in 10% aqueous formaldehyde for macroscopic studies and the other five animals were designated for microscopic analysis. These were arranged in pairs and had the vesicular, prostate, coagulating and bulbourethral glands identified, being structured as mucous glands, which lead into the pelvic urethra. It was concluded that the accessory genital glands found in the paca are the same as those found in most rodents, showing similar histological aspects.
The aim of the study was to show whether it is possible to induce ovulation in the hare by GnRH analogue administration and to carry out an effective artificial insemination (AI). The research was carried out during the breeding and non-breeding season. During the breeding season, plasma progesterone concentrations increased on the 4th day after intramuscular injection of GnRH analogue (buserelin), indicating induced ovulation and corpus luteum development. Prostaglandin F(2)alpha (dinoprost) was an effective luteolytic agent on day 9. During the non-breeding season, the GnRH analogue injection does not cause an increase of progesterone. The 17beta-estradiol concentrations during the breeding and non-breeding season were similar. It was shown that after GnRH analogue administration and artificial insemination with semen diluted in Tris buffer extender 80% females delivered live young (39-43 days after artificial insemination), which proves the effectiveness of inducing ovulation in the hare by means of hormonal stimulation.
The aim of the study was to assess the effect of a different trehalose concentration on the post-thaw viability and fertility of European brown hare spermatozoa. The semen was collected under general anaesthesia with electroejaculation method from 4 males. Immediately after collection, the semen was diluted with an extender of the following composition: Tris 250mM, citric acid 80mM, glucose 70mM, DMSO 1.0M, egg yolk (17%, v/v), and kanamycin (80mg/l)-Protocol I. In Protocols II and III, respectively, 50mM and 100mM of trehalose were added to the extender. Immediately after thawing and after 3h incubation at 37 degrees C, motility characteristics of frozen/thawed semen were assessed with computer-assisted semen analysis system, and a percentage of viable, acrosome intact spermatozoa was evaluated using flow cytometry. Immediately after thawing spermatozoa motility (MOT), average path velocity (VAP), straight velocity (VSL) and curvilinear velocity (VCL) were the highest in the semen frozen without addition of trehalose (P<0.01). After 3h of incubation, MOT and spermatozoa with progressive motility (PMOT) were the lowest in the semen frozen with supplementation of 100mM of trehalose (P<0.01) and VAP, VSL, VCL and amplitude of lateral head displacement (ALH) were significantly lower in the semen frozen with supplementation of 50mM and 100mM of trehalose compared to the semen frozen without the addition of trehalose (P<0.01), which indicates an unfavourable effect of trehalose on the motility characteristics of European brown hare spermatozoa. However, the effect of trehalose on a percentage of viable, acrosome intact spermatozoa was not observed. As a result of artificial insemination, 54.55% females became pregnant after insemination with the semen frozen according to Protocol I, 72.73% and 50% females became pregnant after insemination with the semen supplemented with 50mM and 100mM of trehalose, respectively. The number of young born was 1.67+/-0.52, 1.75+/-1.04 and 1.60+/-0.55, in each group, respectively. There were not any significant differences in the results of artificial insemination between groups. Summing up, it should be stated that the addition of trehalose to the extender did not have a favourable effect on post-thaw viability of European brown hare spermatozoa and an influence of trehalose on the results of artificial insemination was not found, either.
In the last decades, a significant decrease in hare population has been observed; for this reason, the aim of the study was to check if hare semen could be preserved in liquid nitrogen, with an extender used for rabbit semen. The results should provide a basis for creating a gene bank of the species. Ten ejaculates (volume above 0.4 ml, percentage of motile spermatozoa above 75%, spermatozoa concentration above 250 x 10(6) ml), obtained with electroejaculation method from four males, were frozen in an extender of the following composition: Tris (3.028 g), citric acid (1.675 g), glucose (1.25 g), dimethylsulphoxide (DMSO) (4.5%, v/v), egg yolk (17%, v/v) and distilled water to 100.00 ml. The motility of post-thawing spermatozoa was 40.50+/-7.97%, percentage of spermatozoa with normal acrosomes 76.10+/-3.69% and percentage of live spermatozoa 35.05+/-4.21%. Based on the properties of freezing-thawing semen, the hare semen can be successfully preserved in extender used for rabbit semen.
17beta-Hydroxysteroid dehydrogenase type 1 (HSD17B1) is a key enzyme of 17beta-estradiol biosynthesis, which might play an important role in follicular development of the ovary. In this study, we isolated the complete coding sequence of porcine HSD17B1 gene and its unique intron sequences of porcine HSD17B1 gene, identified a single nucleotide polymorphism (SNP: A/C) in intron 4, and developed a PCR-MvaI-RFLP genotyping assay. Association of the SNP and litter size was assessed in two populations (purebred Large White and a experimental synthetic Line (DIV) sows). Statistical analysis demonstrated that, in the first parity, AC animals in experimental synthetic Line (DIV) sows had 0.52 more piglets born compared to the CC animals (P<0.05). In the all parities, pigs with the AA genotype had an additional 1.11 and 0.96 piglets born alive compared to the CC animals (P<0.05) in both experimental synthetic Line (DIV) and purebred Large White, respectively. Experimental synthetic Line (DIV) sows inheriting the AC genotype had additional 0.84 piglets born alive compared to the CC animals (P<0.01) in all parities. In addition, significant additive effect of -0.55+/-0.24 piglets/litter and -0.48+/-0.22 piglets/litter on piglet born alive was detected in both experimental synthetic Line (DIV) sows and purebred Large White lines (P<0.05), respectively. Therefore, HSD17B1 gene was significantly associated with litter size in two populations and could be a useful molecular marker in selection for increasing litter size in pigs.
The objective of the present study was to compare the in vitro production of estradiol-17Beta (E(2)) by cumulus cells in the presence or absence of ovine oocyte. Moreover, the relationship between the concentration of produced estradiol-17Beta and oocyte nuclear maturation was assessed. Ovaries collected from the local abattoir were transported to the laboratory in saline at 30-35 degrees C within 1-3 h after collection. The oocytes of follicles, 2-6 mm in diameter, were recovered by aspiration. The oocytes with evenly granulated cytoplasm and which were surrounded with at least three layers of cumulus cells were selected and subjected to culture in pre-incubated oocyte culture medium (OCM). Before culturing, the selected oocytes were randomly divided into five treatment groups: Group 1, cumulus enclosed oocytes cultured in OCM (Group COCs); Group 2, denuded oocytes cultured in OCM (Group D); Group 3, denuded oocytes co-cultured with a cumulus cell-monolayer in OCM (Group D+M); Group 4, denuded oocytes co-cultured with previously cultured (for 26 h) cumulus cell-monolayer (10(5) cells/ml) in refreshed OCM (Group D+M(26)); Group 5, cumulus cell-monolayer (10(5) cells/ml) cultured in OCM (Group M). After an incubation period (26 h at 38.6 degrees C, 5% CO(2) and 100% humidity), the media were collected and kept at -20 degrees C until hormonal assay. The concentration of E(2) was determined by RIA method. For assessment of nuclear status, the completely denuded oocytes were subjected to DAPI staining. The highest percentage of metaphase II (MII) stage oocytes was observed in Group N (91%) and the lowest percentage was observed in Group D (6%) and Group D+M(26) (6%). The mean production of E(2) was highest and lowest in Group D+M (378.69+/-54.34 pg/ml) and Group D+M(26) (109.15+/-8.24 pg/ml), respectively. The production of E(2) was significantly (P<0.01) higher in Group D+/-M when compared with Groups M and D+/-M(26). Regarding the nuclear maturation, the percentage of MII stage oocytes was significantly (P<0.001) higher in Group COCs compared to the other groups. The results suggest that steroidogenic activity of cumulus cells in in vitro condition can be influenced by the pattern of connection between cumulus cells and the oocyte. Moreover, the nuclear maturation of oocytes is not influenced by the different production levels of E(2).
The objective of this investigation was to develop and evaluate competitive inhibition-enzyme-immunoassays for canine serum oestradiol-17beta (E(2)) and progesterone (P(4)) quantification. Sera from 56 healthy bitches at various stages of oestrus cycle and pregnancy were tested. For E(2) measurement, each sample (0.4 ml) was extracted with diethyl ether and after solvent evaporation the resultant hormone was reconstituted to one-fifth of the original sample volume in aqueous buffer. Each reconstitute (30 microl) was assayed for E(2) to estimate respective serum concentration. For P(4), each sample (10 microl) was directly assayed without extraction. The classic cyclic hormonal pattern during the oestrus cycle of the bitch was observed. The brief, sharp dominance of E(2) during the follicular phase was followed by the long-lasting dominance of P(4) during the luteal phase (late oestrus, dioestrus or pregnancy). During the anoestrus phase both hormones were found at basal levels, with the exception of E(2) during late anoestrus which appeared to be rising. Both assays had acceptable specificity (cross-reactions < or =10%), precision (coefficient of variation (C.V.) < 7%) and accuracy (E(2) recovery: 97%; P(4) recovery: 104.7%). The sensitivity of E(2) and P(4) assay was 4 pgml(-1) and 0.28 ngml(-1), respectively.
The role of estradiol-17beta on nuclear and cytoplasmic maturation of pig oocytes was investigated in the present study. To determine the estradiol effect, oocytes were cultured for 42 h in a steroid free medium composed of mTCM-199 supplemented with LH, FSH and 10% charcoal extracted follicular fluid. Estradiol receptor (ER), detected by a binding assay, were present in cumulus cells and oocytes during maturation with higher levels observed at 24 h of culture in the oocytes and at 36 h in the cumulus cells. To block estradiol action an antiestrogen (1-p-dimethylaminoethoxyphenyl-1,2-diphenyl-1-butene (tamoxifen)) was added to the maturation medium at various concentrations. The percentage of treated oocytes that underwent nuclear maturation was similar (P>0.05) to the control group. Cytoplasmic maturation, determined by the ability to form female pronucleus (FPN) and male pronucleus (MPN), was not different (P>0.05) among all groups. The presence of 4-hydroxy-4-androstene-3-17-dione (4-OHA) also did not influence nuclear (P>0.05) or cytoplasmic maturation (P>0.05). The results suggest that estradiol is not involved in maturation of pig oocytes. However, the present experiment used pronuclei formation as the endpoint, no studies were done in regard to estradiol's effects on the embryonic development.
Plasma luteinizing hormone (LH) and testosterone (T) levels in three normal male Beagles increased markedly, the LH levels peaking at 30 or 45 min and the T levels at 45 or 60 min respectively, after a subcutaneous injection of 1 microgram kg-1 gonadotropin releasing hormone agonist (GnRH-A). Two Collies and a Great dane diagnosed as oligozoospermic and two Shetland sheep dogs diagnosed as azoospermic by evaluation of semen quality were treated with 1 microgram kg-1 GnRH-A after blood collection. Their plasma levels of LH, T and estradiol-17 beta (E2) were measured by radioimmunoassay for the purpose of investigating the effect of hormone therapy on spermatogenic dysfunction and the mechanism on improvement of semen quality. The semen quality of one of the Collies had improved 4 weeks after the GnRH-A treatment. The dog was treated with GnRH-A again and mated with a bitch 4 days later. The bitch gave birth to five puppies. The other dogs, whose semen quality had not improved, were treated with an intramuscular injection of 500 or 1000 IU human chorionic gonadotrphin (hCG) per animal. Since the semen quality of the other Collie and the Great Dane improved temporarily 2 and 4 weeks, respectively, after hCG treatment, the former was mated with a bitch 5 days later. The bitch gave birth to a litter of seven puppies. These hormone treatments, however, had no effect on the azoospermia in the two Shetland sheep dogs. Although the mean plasma LH and T levels in the dogs with oligozoospermia had been low, their LH levels gradually increased after hormone treatment. There were no marked changes in plasma T or E2 levels. These findings indicate that the semen quality of dogs with oligozoospermia can be temporarily improved between 2 and 4 weeks after a single injection of GnRH-A or hCG and the fertility of the dogs restored by the injection.
Although the mammary gland of many species secretes estradiol (E(2)), nothing is known of E(2) secretion in the porcine gland. The present study was designed to investigate whether porcine mammary gland was a source of E(2), and to test the influence of individual and combined effects of exogenous progesterone and estradiol benzoate (EB) on the secretion of E(2). Immature crossbred gilts were ovariectomized at 7 months of age followed by 4 weeks later by steroid hormone replacement therapy to produce estradiol and progesterone (P(4)) blood concentrations similar to those observed during a normal estrous cycle. Arterial and venous blood plasma (from carotid artery and anterior mammary vein, respectively) were sampled for 2h at 10 min intervals. Plasma concentrations of progesterone, androstenedione (A(4)), testosterone (T), estrone (E(1)) and estradiol were determined by RIA. In all gilts treated with progesterone alone or in combination with EB, concentrations of P(4), A(4) and E(1) in blood collected from venous outflow were lower compared to concentrations in arterial blood, whereas concentrations of E(2) were higher in blood plasma from the anterior mammary vein compared to plasma from the carotid artery. The results indicated that the porcine mammary gland secreted E(2). Increased concentrations of plasma E(2) collected only from P(4)-treated animals suggested that progesterone activated enzymes involved in steroidogenesis in porcine mammary gland, or those utilized in its metabolism.
The effect of 17beta-oestradiol and oestrous stage-specific cow serum on bovine oviductal epithelial cell monolayers to extend the viability of co-cultured bull spermatozoa was examined. Monolayers of cells from ampullary and isthmic segments were pre-treated with medium containing either oestrous cow serum, luteal-phase cow serum, 1 microg/ml 17beta-oestradiol + foetal bovine serum or foetal bovine serum alone (control) before the addition of motile frozen/thawed spermatozoa. Motility was visually assessed throughout a 48 h co-incubation period, while fertilising ability of spermatozoa was evaluated by adding in vitro matured bovine oocytes. Pre-treatment with 17beta-oestradiol or oestrous cow serum resulted in a higher percentage of motile spermatozoa after 18 h in isthmic and after 36 h in ampullary cultures compared with the control, but pre-treatment did not affect fertilisation rates. Only at 42 h in ampullary cultures was motility higher in luteal serum pre-treated cultures compared to the control. Motility was also assessed in medium conditioned by pre-treated monolayers. Pre-treatment with 17beta-oestradiol enhanced the ability of conditioned medium to prolong motility and medium conditioned with oestrous cow serum was superior to medium conditioned by luteal-phase serum at maintaining motility. In conclusion, the ability of oviductal epithelium to prolong the motility of spermatozoa is enhanced by 17beta-oestradiol.
Plasma FSH, LH, estradiol (E2) and progesterone (P4) profiles and patterns of follicular growth and regression by ultrasonography were determined after E2 treatment (1 microg/kg) in anestrous ewes. Fifteen ewes were treated with one (group I, n=7) or two (group II, n=4) i.m. injections of E2 with a 24h interval, or two oil injections with a 24h interval (group C, n=4). Blood samples for E2, P4, FSH and LH determinations were collected daily 4 days before the initiation of the treatment (day 0), when bleeding increased to every 2h starting 2h before treatment until 56h after the first injection and from then on every 6h until day 8, and twice per day till the end of the experiment (day 9). During the experimental period (days -4 to 9), transrectal ultrasonic examinations were carried out daily using a 7.5 MHz linear array probe. Number and size of follicles > or =3mm in diameter were recorded. No estrous was detected before, during or after treatment. LH and FSH surges were observed 10-18h after the first E2 injection. The second E2 injection stimulated another release of LH but no surges. E2 inhibited FSH levels before the surge and the second E2 injection induced a longer inhibition. No ovulation was detected by ultrasonography during the experimental period and P4 levels remained low (<0.7 nmol/l) before, during and after the treatment in all ewes. There was an effect of E2 treatment on the diameter of the largest follicle, a decrease could be observed 3 days after the first injection in both ewes of groups I and II. The E2-treated groups had a higher frequency of ewes showing wave emergence on day 3 (day 1.5+/-1,2.4+/-0.4 and 2.5+/-0.5 for control, groups I and II). LH and FSH surges were observed after E2 treatment, but were not able to provoke ovulation neither luteinization. In contrast, the treatment was associated with the regression of the largest follicle and with emergence of a new follicular wave on day 3.