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.
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 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.
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).
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) 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.
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.
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.
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.
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.
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.