[Show abstract][Hide abstract] ABSTRACT: The aim of the study was to evaluate whether resveratrol supplementation of bovine culture medium improves in vitro blastocyst development, embryo cryotolerance and cell numbers. Abattoir-derived oocytes were matured and fertilized in vitro according to standard procedure. Twenty hours after IVF, zygotes were cultured in SOF medium, supplemented with 0 (control, n = 439), 0.25 μM (n = 422), 0.5 μM (n = 447) and 1 μM resveratrol (n = 416). On Day 7 (IVF = Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5 M sucrose. Development rate, i.e. the percentage of embryos resuming development to reach a more advanced stage, and hatching rate were evaluated after 24 and 48 h culture. Blastocysts cultured with (0.5 μM) and without resveratrol underwent differential staining to count inner cell mass (ICM) and trophectoderm (TE) cells. Resveratrol during culture did not increase blastocyst yields (57.1, 57.7, 59.2 and 46.6%, respectively in 0, 0.25, 0.5 and 1 μM resveratrol). However, 0.5 μM resveratrol improved embryo cryotolerance compared to the control, as indicated by higher development rates (67.3% vs 50.3%, respectively; P < 0.01) and hatching rates (58.9% vs 30.9%, respectively; P < 0.01) recorded after 48 h post-warming culture. Blastocysts produced in the control and in 0.5 μM resveratrol groups had similar numbers of ICM (34.1 and 36.4, respectively), TE (88.1 and 85.3, respectively) and total (122.2 and 121.7, respectively) cells. In conclusion, low levels of resveratrol during in vitro culture improve the quality of IVP bovine embryos, as indicated by their increased resistance to cryopreservation.
Full-text · Article · Oct 2014 · Animal Reproduction Science
[Show abstract][Hide abstract] ABSTRACT: Abstract Text: Dairy Buffalo farming has a relevant economic importance in Italy, mainly due to the high value of the most important dairy product, the mozzarella. Fat is a major component of buffalo dairy products. Its consumption can provide great opportunities for increasing the intake of fatty acids (FA) with potential health properties, especially as far as CLA isomers and omega-3 FA are concerned. Therefore the purpose of this survey was to investigate the variation in FA profile of milk fat of Mediterranean water buffalo fed a total mixed ratio composed mainly by corn silage and pelleted concentrate. Milk samples from 398 Italian Water buffaloes farmed in 18 herds located in Campania were collected at different months of production. FA composition of milk samples was determined by GC. Data were analyzed using a linear model, that includes age, days in milk (DIM) and month of calving as fixed effects and herd as a random effect. Saturated FA (SFA) represented 71.6% of total FA (ranging from 57.9% to 85.9%) and C16:0 and C18:0 were the most represented (34.8% and 11.1%, respectively). Monounsaturated FA (MUFA) were 25.17% of total milk FA (ranging from 12.6% to 37.4%) and C18:1c9 represent the majority (76% of total MUFA). The concentration of polyunsaturated FA (PUFA) was 3.21% of total FA ranging from 1.39% to 5.11%. with C18:2 n6 predominating (49% of total PUFA) while C18:3 n3 was present in a lower amount 0.32% of total FA. Trans FA (TFA) represented 1.70% of total FA with C18:1 t11 the most abundant (59% of total TFA). CLA isomers amounted to 0.76% of total milk FA and the isomer c9, t11 CLA represent the majority (55% of total CLA). Statistical analysis showed that milk FA were not significantly influenced by the age of animal, except for TFA content (P<0.05) that was higher in younger animals (1.94%). Month of calving significantly influenced FA composition of buffalo milk only for total CLA and TFA (P<0.05), evidencing a seasonality effect o these traits. DIM affected significantly all the group of FA analyzed (P<0.01) denoting a marked lactation curve effects. The FA profile obtained in this study is typical of animals farmed in intensive systems, with a reduced occurrence of unsaturated fatty acids, compared to graze-based systems.
Keywords: Buffalo milk, fatty acid profile
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to determine the effect of Methyl-B-Cyclodextrin (MBCD) on capacitation of buffalo sperm. Frozen/thawed semen was incubated in the absence of capacitating agents (negative control), in the presence of 10 µg/ml heparin (positive control) and of 1, 2, 4 and 8 mg/ml MBCD for 2 and 4h. At each incubation time, sperm motility was evaluated by phase contrast microscopy. Capacitation was assessed by the sperm ability to undergo acrosome reaction after lysophosphatidylcholine treatment, evaluated with viability by Trypan blue-Giemsa. After 2 h capacitation increased (P<0.01) in MBCD groups (39.2±1.4, 44.5±3.3, 56.7±1.5 and 62.5±3.8, with 1, 2, 4 and 8 mg/ml MBCD, respectively) compared to the negative and positive controls (27.5±1.0 and 28.0±0.8, respectively). Likewise, after 4 h the percentage of live capacitated sperm was higher at increasing concentration of MBCD (31.0±0.7, 34.5±1.7, 42.0±1.9, 49.2±2.8, 62.3±1.5 and 70.8±1.7 in negative control, positive control and with 1, 2, 4 and 8 mg/ml MBCD, respectively; P<0.01). After 2 h sperm motility was lower (P<0.01) in 4 and 8 mg/ml MBCD groups (43.3±2.1 and 25.0±3.2, respectively) than in negative control, positive control, 1 and 2 mg/ml MBCD groups (55.0±1.8, 48.3±2.8, 61.7±2.8, 56.7±1.1, respectively). After 4 h the lowest sperm motility was observed with higher MBCD concentrations (40.0±0.0, 46.7±4.2, 51.7±4.6, 50.0±0.0, 40.0±3.7 and 6.7±1.1, in negative control, positive control, 1, 2, 4 and 8 mg/ml MBCD, respectively; P<0.01). In conclusion, MBCD improved sperm capacitation in a dose-dependent manner while decreasing the sperm motility at higher concentrations.
[Show abstract][Hide abstract] ABSTRACT: The transcriptome profiles were compared for buffalo embryos with normal growth and embryos with retarded growth on Day 25 after mating. Embryos with retarded growth on Day 25 after mating have a reduced likelihood of undergoing attachment to the uterine endometrium and establishing a pregnancy. Italian Mediterranean buffaloes were mated by AI and on Day 25 underwent trans-rectal ultrasonography to ascertain embryo development. Embryos with an embryonic width (EW)>2.7 mm were classed as normal embryos and embryos with an EW<2.7 mm were classed as retarded embryos. Three buffaloes with embryos of the largest EW (3.7, 3.7 and 3.9 mm) and three buffaloes with embryos of the smallest EW (1.5, 1.6 and 1.9 mm) were slaughtered on Day 27 to recover embryos for transcriptome analysis using a bovine custom designed oligo array. A total of 1,047 transcripts were differentially expressed between embryos with normal growth and embryos with retarded growth. Retarded embryos showed 773/1,047 (74%) transcripts that were down-regulated and 274/1,047 (26%) transcripts that were up-regulated relative to normal embryos; in silico analyses focused on 680/1,047 (65%) of the differentially expressed transcripts. The most altered transcripts observed in retarded embryos were associated with membrane structure and function and with metabolic and homeostasis maintenance functions. Other notable functions altered in retarded embryos were developmental processes and in particular nervous system differentiation and function. Specific biochemical pathways such as the complement cascade and coagulation were also altered in retarded embryos. It was concluded from the findings that buffalo embryos with retarded growth on Day 25 after mating show altered gene expression compared with normal embryos, and some de-regulated functions are associated with attachment to the uterine endometrium.
[Show abstract][Hide abstract] ABSTRACT: The aim of this research was to estimate the variability between buffalo as oocyte donors. In Experiment 1, reproductive variables were retrospectively analyzed in buffalo (n=40) that underwent repeated ovum pick up (OPU), over 16 puncture sessions (PS). The follicular recruitment among individuals and the relationship between follicular population and oocyte production were evaluated. In Experiment 2, eight buffalo underwent OPU for 28 PS and the oocytes were processed separately to correlate follicular and oocyte population at the first PS to blastocyst (BL) production. In Experiment 1, the average number of total follicles (TFL), small follicles (SFL), cumulus-oocyte complexes (COC) and Grade A+B COC recorded in each 4-PS period had great repeatability (r=0.52, 0.54, 0.60 and 0.57, respectively). The average number of Grade A+B COC recovered during the subsequent 15 PS was positively correlated with the first PS number of TFL (r=0.60; P<0.001), SFL (r=0.68; P<0.001), COC (r=0.48; P<0.01) and Grade A+B COC (r=0.40; P<0.05). In Experiment 2, a large variability among animals was observed in blastocyst yields. When animals were grouped according to the BL yield, the greatest BL yield group had a greater (P<0.05) number of TFL (8.3±0.9 compared with 5.6±0.7) and SFL (7.3±0.3 compared with 3.8±0.7) at the first PS than the lesser BL yield group. The average number of BL produced over the subsequent sessions was correlated with the number of TFL (r=0.80; P<0.05) and COC (r=0.76; P<0.05) observed at the first PS. These results demonstrated a donor influence on the oocyte and BL production, suggesting a preliminary screening to select the donors with greater potential.
Full-text · Article · Dec 2013 · Animal reproduction science
[Show abstract][Hide abstract] ABSTRACT: Brucellosis is considered the world's most widespread zoonotic infection. It causes abortion and sterility in livestock leading to serious economic losses and has even more serious medical impact in humans, since it can be a trigger to more than 500,000 infections per year worldwide. The aim of this study was to evaluate the role of Haematopinus tuberculatus, a louse that can parasitize several ruminants, as a new host of brucellosis. Louse specimens were collected from seropositive and seronegative water buffaloes and divided in 3 developmental stages: adults, nymphs and nits. All samples were separately screened for Brucella spp. DNA and RNA detection by Real Time PCR. In particular, primers and probes potentially targeting the 16S rRNA and the Brucella Cell Surface 31kDalton Protein (bcsp31) genes were used for Real Time PCR and buffalo beta actin was used as a housekeeping gene to quantify host DNA in the sample. A known amount of B. abortus purified DNA was utilized for standard curve preparation and the target DNA amount was divided by the housekeeping gene amount to obtain a normalized target value. A further molecular characterization was performed for Brucella strain typing and genotyping by the Bruce-ladder, AMOS-PCR and MLVA assays. Data were statistically analysed by ANOVA.
Brucella abortus DNA and RNA were detected in all developmental stages of the louse, suggesting the presence of viable bacteria. Data obtained by MLVA characterization support this finding, since the strains present in animals and the relative parasites were not always identical, suggesting bacterial replication. Furthermore, the detection of Brucella DNA and RNA in nits samples demonstrate, for the first time, a trans-ovarial transmission of the bacterium into the louse.
These findings identified H. tuberculatus as a new host of brucellosis. Further studies are needed to establish the role of this louse in the epidemiology of the disease, such as vector or reservoir.
Full-text · Article · Dec 2013 · BMC Veterinary Research
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate whether the developmental speed affects the cryotolerance of in vitro produced buffalo embryos. In Experiment 1, abattoir-derived oocytes were in vitro matured, fertilized and cultured. The embryos produced by Say 7 of culture were vitrified at the tight morula (TM), early blastocyst (EBL), blastocyst (BL), expanded-blastocyst (XBL) and hatched-blastocyst (HBL) stage. The embryos were vitrified by cryotop in 16.5% ethylene glycol (EG) and 16.5% dimethyl sulfoxide (DMSO) and 0.5 M sucrose. Embryos were warmed in 0.25 M sucrose for 1 min and then in 0.15 M sucrose for 5 min and cultured in vitro for 24 h, to evaluate post-culture viability. In Experiment 2, ovum pick-up (OPU) was carried out on lactating buffaloes to produce embryos that were vitrified-warmed and transferred into synchronized recipients. The lowest (P< 0.01) survival rates were recorded with TM (22.4%) and the highest (P< 0.01) with HBL (84.5%), whereas intermediate results were observed with EBL, BL and XBL (54.5, 64.7, 67.9%, respectively). Pregnancy rate on both Days 25 and 45 was significantly higher (P< 0.05) for faster ( XBL and HBL) developing embryos (75.0% and 62.5%, respectively) than for the slower (TM, EBL and BL) developing counterparts (36.4% and 0, respectively). Interestingly, pregnancies to term were only recorded when HBL were transferred (20%). In conclusion, it was demonstrated that the chronology of development is a major factor affecting the cryotolerance of in vitro produced buffalo embryos.
Full-text · Article · Oct 2013 · Italian Journal of Animal Science
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to compare the proteome profiles of the chorioamnion and corresponding caruncle for buffalo embryos that had either normal or retarded development on Day 25 after artificial insemination (AI). In experiment 1, embryos that were to subsequently undergo late embryonic mortality had a lesser width on Day 25 after AI than embryos associated with pregnancy on Day 45 after AI. In experiment 2, 25 Italian Mediterranean buffaloes underwent transrectal ultrasonography on Day 25 after AI and pregnant animals were categorized as two groups based on embryonic width: normal embryos (embryonic width > 2.7 mm) and retarded embryos (embryonic width < 2.7 mm). Three buffaloes of each group were slaughtered on Day 27 after AI to collect chorioamnion and caruncle tissues for subsequent proteomic analyses. Two-dimensional difference gel electrophoresis (2D-DIGE) and MALDI-ToF/ToF mass spectrometer analysis were used to ascertain the proteomic profiles. To confirm 2D-DIGE-results, three selected proteins were analyzed by Western blot. The proteomic profiles of the chorioamnion of retarded embryos and the corresponding caruncles showed differences in the expression of several proteins compared to normal embryos. In particular, a down-regulation was observed for proteins involved in protein folding (HSP 90-alpha, calreticulin), calcium binding (annexin A1, annexin A2), and coagulation (fibrinogen alpha-chain) (P < 0.05) whereas proteins involved in protease inhibition (alpha-1-antiproteinase, serpin H1, serpin A3-8), DNA and RNA binding (heterogeneous nuclear ribonucleoproteins A2/B1 and K), chromosome segregation (serine/threonine-protein phosphatase 2A), cytoskeletal organization (ezrin), cell redox homeostasis (amine oxidase-A), and hemoglobin binding (haptoglobin) were up-regulated (P < 0.05).
Full-text · Article · Apr 2013 · Biology of Reproduction
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to make a comparison among the lactations of the buffalo cows that were both members of the Herd Book and under Milk Recordings in order to identify some parameters useful in the planning of milk production and then to improve the deseasonalization of the quantity of produced milk. The milk production primarily depends by the trend of the lactation curve, and the factors which composed it are: peak efficiency and persistence. The previous parameters are the key in the description of the model of the release of milk, particularly: the peak efficiency is the maximum production during the lactation, and the persistence describes the magnitude of the decline in production. The Wood model is the most widely used function for the description of lactation curves because it provide to extrapolate a lot of information relatives to the curve when the data are not available. The Wood model also helps the breeders in the management of the herd, in fact a rational management would implies to estimate the amount of milk that will be produced. The work presented below has availed of the Wood mathematical model (1967) and has established that it is possible to develop a forward estimate of the lactation curve with a good approximation of what will be the future lactations.
No preview · Article · Jan 2013 · Buffalo Bulletin
[Show abstract][Hide abstract] ABSTRACT: The aim was to ascertain whether relationships between corpus luteum (CL) vascularization, CL function, and pregnancy outcome in AI in buffaloes were consistent across the breeding season and transition period to the nonbreeding season in a Mediterranean environment. Stage of the estrous cycle in Italian Mediterranean buffaloes was synchronized using the Ovsynch with timed AI program and buffaloes were mated by AI in both the breeding season (N = 131) and transition period (N = 125). Detailed investigation of CL structure and function was undertaken in 39 buffaloes at each of the respective times using realtime B-mode/color-Doppler ultrasonography on Days 10 and 20 after AI. Progesterone (P4) concentrations were determined by RIA in all buffaloes. Pregnancy rate on Day 45 after AI was greater (P < 0.05) during the breeding season (58.0%) than the transitional period (45.6%) and this was primarily the result of a lower (P < 0.05) late embryonic mortality during the breeding season (7.3%) compared with the transition period (23%). Circulating concentrations of P4 on Days 10 and 20 after AI were greater (P < 0.01) during the breeding season (4.6 ± 0.3 and 3.4 ± 0.2, respectively) than during the transition period (1.6 ± 0.12 and 1.8 ± 0.2, respectively), and this was independent of reproductive status as there was no interaction between pregnancy and season. Corpus luteum time average medium velocity at Day 10 after AI was greater (P < 0.01) during the breeding season (19.3 ± 1.5) than in the transitional period (8.3 ± 0.7). There were positive correlations in pregnant buffaloes between CL time average medium velocity and P4 concentrations on Day 10 (r = 0.722; P < 0.01) and Day 20 (r = 0.446; P < 0.01) after AI. The findings were interpreted to indicate that relationships between CL vascularization, CL function, and pregnancy outcome in AI in buffaloes are consistent across the breeding season and transition period to the nonbreeding season. The distinction between the breeding season and the transition period is the relatively low proportion of buffaloes that have CL function and P4 concentrations required to establish a pregnancy during the transition period, which is manifested in a greater incidence of embryonic mortality.
[Show abstract][Hide abstract] ABSTRACT: This study was designed to evaluate the effect of season on in vivo oocyte recovery and embryo production in Mediterranean Italian buffalo (Bubalus bubalis). For this purpose repeated transvaginal ultrasound-guided ovum pick up (OPU) was conducted twice a week throughout autumn, mid-winter (transitional period) and spring-summer. The number and size of follicles was determined before puncture. The recovered oocytes were first classified in morphological categories and then used for in vitro embryo production (IVEP) according to standard procedures. The mean number of total follicles observed per session did not differ among the three periods we examined (on average 4.6). Although season did not considerably affect the number of oocytes recovered (on average 2.3/buffalo/session), the number of degenerated and abnormally expanded oocytes increased during autumn. Furthermore, the percentage of abnormally expanded oocytes significantly increased during autumn (6.1%) compared with both the transitional period and spring-summer (1.9 and 2.3%, respectively). Interestingly, the embryo output we recorded at day 7, in terms of tight morulae-blastocysts was higher in autumn (30.9%) compared to the other two periods (13.3% and 10.3%, respectively, in spring-summer and in the transitional period; P<0.01). The results of this trial demonstrated that the morphological features of the oocytes did not vary substantially among the considered periods, with the exception of degenerated and abnormally expanded oocytes. On the other hand, the oocyte developmental competence improved in autumn compared to spring-summer and the transitional period. This datum reflects buffalo reproductive pattern expressed in vivo at Italian latitudes.
[Show abstract][Hide abstract] ABSTRACT: The aim of this work was to evaluate whether minimizing the glucose concentration during culture or replacing the hexose with other energy substrates and/or embryotrophic compounds would affect the in vitro development, the resistance to cryopreservation and the sex ratio of bovine embryos. In vitro matured and fertilized oocytes were randomly assigned to 4 groups for in vitro culture, that differed in the energy substrates included: group A) 1.5 mM glucose, as in standard SOF; group B) 0.15 mM glucose; group C) 0.125 mM G3P, in the presence of 0.15 mM glucose and group D) 0.34 mM citrate, in combination with 2.77 mM myo-inositol. Blastocysts were evaluated on day 7, then vitrified by cryotop in 16.5% DMSO, 16.5% EG and 0.5 M sucrose and warmed in decreasing concentration of sucrose (0.25 to 0.15 M sucrose). The survival rates were assessed after 24 h in vitro culture. Finally, the blastocysts produced were sexed by PCR. An increased blastocyst rate was recorded in groups B, C and D, i.e., when glucose concentration was reduced, compared to group A (28.2, 41.0, 35.7 and 35.8, respectively in groups A, B, C and D; P < 0.01). However, the embryos cultured in group D showed the slowest developmental speed, indicated by the lowest percentage of advanced stage-embryos (expanded and hatched blastocysts) out of the total blastocysts (56.1, 45.8, 56.9 and 31.8 %, respectively in groups A, B, C and D; P < 0.01). Furthermore, survival rates after 24 h culture of vitrified-warmed blastocysts also decreased in group D (73.3, 73.1, 71.4 and 58.4%, respectively in groups A, B, C and D; P < 0.01). Interestingly, in group D a higher percentage of female embryos was obtained compared to group A, with intermediate values in groups B and C (45.6, 53.4, 50.0 and 61.5%, respectively in groups A, B, C and D; P < 0.05). In conclusion, it was demonstrated that the energy substrate during in vitro culture affects both the production and the viability of blastocysts. Furthermore, manipulating the metabolic profile of embryos during in vitro culture may have an impact on sex ratio.
[Show abstract][Hide abstract] ABSTRACT: The objectives of this work were to evaluate whether the sperm penetration speed is correlated to the in vitro fertility and whether adapting the gamete co-incubation length to the kinetics of the bull improves in vitro fertility and affects the sex ratio. In vitro matured oocytes were co-incubated with spermatozoa from four different bulls (A-D). At various post-insemination (p.i.) times (4, 8, 12, 16 and 20 h), samples of oocytes were fixed and stained with DAPI for nuclei examination, while the remaining ones were transferred into culture to evaluate embryo development. The blastocysts produced were sexed by PCR. Two bulls (A and B) had faster kinetics than the others (C and D), as shown by the higher penetration rates recorded at 4 h p.i. (43%, 30%, 11% and 6%, respectively for bulls A, B, C and D; p<0.01). The differences in the kinetics among bulls did not reflect their in vitro fertility. The incidence of polyspermy was higher for faster penetrating bulls (36%, 24%, 16% and 4%, respectively for bulls A, B, C and D; p<0.01) and at longer co-incubation times (0%, 16%, 19%, 30% and 34%, respectively at 4, 8, 12, 16 and 20 h p.i.; p<0.01). The fertilizing ability of individual bulls may be improved by adapting the co-incubation length to their penetration speed. A sperm-oocyte co-incubation length of 8 h ensured the greatest blastocyst yields for the two faster penetrating bulls. On the contrary, 16 h co-incubation was required to increase (p<0.01) cleavage rate of the two slower bulls. Bulls with a faster kinetics did not alter the embryo sex ratio towards males. The female/male (F/M) ratios recorded were 2.1, 1.4, 1.2, 1.3 and 1.6, respectively at 4, 8, 12, 16 and 20 h p.i.
[Show abstract][Hide abstract] ABSTRACT: The use of sexed semen in farm animal production and genetic improvement has been shown to be feasible with variable degree of efficiency in a number of species, and proved to be economically viable in cattle. In the last two decades, various newly developed reproductive technologies applicable in buffaloes have mushroomed. Recently, following the birth of the first buffalo calves using AI with sexed semen, commercial interest to exploit sexing of semen in this species too is aroused. In order to verify the successful adoption of this technology in the buffalo, the present study on the use of sexed semen for AI was carried out and compared with conventional artificial insemination using nonsexed semen. A total of 379 buffalo heifers were used for synchronization of ovulation using the Presynch protocol in the South of Italy. Selected animals at the time of AI were randomly allocated to three different experiment groups: (1) 102 animals subjected to AI in the body of the uterus with sexed semen (SS body); (2) 104 animals subjected to AI in the horn of the uterus with sexed semen (SS horn); and (3) 106 animals subjected to AI in the body of the uterus with conventional nonsexed semen (NSS body). Semen of three buffalo bulls was sexed by a collaborating company and commercially distributed in 0.25 mL straws with a total of 2 million sexed spermatozoa. Pregnancy rates were first assessed at Day 28 following AI, and rechecked at Day 45 by ultrasound. Pregnancy rates were nonsignificantly different between animals inseminated with sexed or nonsexed semen: 80/206 (38.8%) and 40/106 (37.7%), respectively (P = 0.85). However, site of insemination of sexed semen affected pregnancy rate significantly as higher pregnancy rates were obtained when sexed semen was deposited into the body rather than the horn of the uterus: 46/101 (45.5%) and 34/105 (32.3%), respectively (P = 0.05). In conclusion, the use of sexed semen in buffalo heifers gave satisfactory and similar pregnancy rates when compared with conventional nonsexed semen. Deposition of sexed semen into the body of the uterus, however, increased pregnancy rates significantly.
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the effect of an Ovum Pick-up (OPU) treatment carried out for 9 months in buffalo (Bubalus bubalis) species. Eight pluriparous non-lactating buffalo cows underwent OPU for 9 months. Recovered cumulus enclosed oocytes (COCs) were classified and COCs suitable for in vitro embryo production (IVEP) were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the blastocyst (Bl) stage. Animals were monitored for a total period of 270 days, but at the summer solstice, follicular turnover decreased and at the 68-day of the trial, we decided to increase the OPU sampling interval from 3-4 to 7 days. It was therefore possible to distinguish two phases: a first phase (18 sessions), during which OPU was carried out twice weekly and a second phase (16 sessions) during which OPU sessions were performed weekly. This reduction did not modify the percentage of good quality COCs, while the incidence of grade D COCs decreased (P<0.01). Furthermore, embryo production was higher in the second phase, either if embryos were calculated on the total recovered COCs (8.3% vs. 21.4%; P<0.01) and on grade A+B COCs (13.0% vs. 32.1%; P<0.01), that supposedly should have given similar blastocyst yield. During the total period of the trial it was possible to distinguish a first period of 6 months (34 sessions) characterized by blastocyst production (0.36 blastocyst/buffalo/session), followed by an unproductive period of 3 months (12 sessions), during which embryos were not produced. During the first 6 months a higher (P<0.01) number of follicles (5.06 vs. 3.71), small follicles (3.38 vs. 2.07), total COCs (2.58 vs. 1.56) and good quality (A+B) COCs (1.51 vs. 0.94) per subject/session were recorded compared to the last 3 months. No Blastocyst were produced during the second period, even if the percentage of grade A+B COCs was similar to that recorded during the first period. In conclusion, buffalo cows submitted to repeated OPU sampling for a 9-month period, showed a decline of follicle recruitment and oocyte collection after the first two months of samplings. After 6-month of samplings, in spite of the quality grade of the collected oocytes, we found a drop in their developmental competence.
Full-text · Article · Feb 2011 · Animal reproduction science
[Show abstract][Hide abstract] ABSTRACT: At Italian latitudes, buffalo (Bubalus bubalis) is a seasonally polyestrous species, showing an improved reproductive efficiency when daylight decreases (autumn). The aim of the present study was to evaluate the influence of the season on buffalo oocyte recovery rate, on oocyte quality, assessed on morphological basis, and developmental competence after in vitro fertilization. For this purpose, buffalo ovaries were collected from a local abattoir and the oocytes obtained by aspirating the follicles were evaluated, classified and, if considered of good quality, devolved to the different procedures of IVEP. In general, no differences were found in terms of oocyte recovery per ovary among seasons, but interestingly, the percentage of small oocytes was higher (P<0.05) during spring and summer (0.9±0.1 and 0.9±0.2) compared to autumn and winter (0.3±0.1 and 0.2±0.1). Both cleavage and embryo rate increased during the period from October to December (71.7±3.1 and 26.5±2.1, respectively) compared to the period from April to June (58.0±2.4 and 18.8±1.6, respectively), thus reflecting the in vivo reproductive behavior. Nevertheless, it is worth emphasizing that transferrable embryos were produced in vitro, even during the unfavorable season, but with decreased efficiency. In conclusion, these results suggest to avoid the oocyte collection during spring when planning OPU trials in order to save resources and improve the benefits/costs ratio.
Full-text · Article · Jan 2011 · Animal reproduction science
[Show abstract][Hide abstract] ABSTRACT: The purpose of the review is to describe the factors that affect fertility in domestic water buffalo (Bubalus bubalis) and the techniques that enable an improvement in reproductive performance. On Italian and Latin American farms where natural mating is practiced and bulls are always present in the herd, the inter-calving interval is approximately 400 days and the culling rate is lower than 15%. The buffalo has a tendency for seasonal reproductive activity. Reproduction is favoured when there is a decrease in day length. Ovarian activity stops if conception does not occur within 3 to 5 ovarian cycles. It is important, therefore, that appropriate management of the transition period is practiced, particularly with respect to the hygienic conditions of the uterus. In tropical countries located north of the equator, feed deficiencies and heat stress are considered the main factors that lead to poor fertility in the summer. In Pakistan, for example, the increase in body condition score during the autumn was associated with the commencement of the breeding season in buffaloes. Anoestrus is observed also in Italy, however, where the average daily temperature during the same period is 13.5 to 23.5 degrees C and feeding is constant throughout the year. The only common element between the two areas is the progressive increase in daylight hours between April and June and the day length greater than 12 hours up to September. In Italian herds that apply an out-of-season breeding strategy, an improvement in fertility (measured as the percentage of corpora lutea corresponding to subsequent pregnancy) is observed when water pools are present on the farm. This demonstrates that an improvement in environmental conditions reduces the incidence of embryonic mortality and/or abnormal cycles. To summarize, in the absence of serious nutritional problems, an improvement in environmental conditions increases fertility in buffalo.
[Show abstract][Hide abstract] ABSTRACT: The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control. An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01). In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.
[Show abstract][Hide abstract] ABSTRACT: This review brings together information on ovarian physiology in buffaloes including folliculogenesis, ovulation, and the development and function of the corpus luteum. Features of embryonic development are also considered. The buffalo is classified as a short-day breeder but in equatorial zones can show oestrous cycles throughout the year provided that nutrition is adequate to maintain reproductive function. In sub-tropical zones and at higher latitudes, day length is often the major determinant of reproductive function including the occurrence of regular oestrous cycles, duration of oestrus, and the period to resumption of ovulation postpartum. Indeed, at higher latitudes buffaloes that give birth during the period of increasing day length may not show a resumption of ovulation until the following period of decreasing day length. This can have a major impact on the productive value of buffaloes and requires the development and utilisation of practical and effective assisted breeding technology for out-of-season breeding in buffaloes. Embryonic development in buffaloes occurs at a faster rate than in cattle and this has implications for the earlier establishment and functionality of the corpus luteum in buffaloes. It would appear that the interrelationships between the development of the early conceptus, corpus luteum function, uterine preparation, and maternal recognition of pregnancy, are more closely time-bound in buffaloes compared with cattle. The phase of embryonic attachment would seem to be a critical period for determining the reproductive outcome in buffaloes.
Full-text · Article · Aug 2010 · Animal reproduction science
[Show abstract][Hide abstract] ABSTRACT: The aim of this study was to evaluate the effect of exogenous progesterone supplementation on superovulatory response in buffaloes that has undergone a multiple ovulation program. Fourteen Mediterranean buffaloes were divided into two groups and received a 4-day decreasing dosage of an equal mixture of 500 IU of FSH and LH starting on day 8 of the cycle. In group A (n = 7) a progesterone-releasing intravaginal device was removed on day 8, whereas in group B (n = 7) it was left till day 10, when PGF2alpha was administered. Eighty hours later, buffaloes were artificially inseminated and after 6 days they undergone uterine flushing. A higher (P < 0.05) number of corpora lutea (8.3 vs. 5.7) and embryo/flushing/buffalo (2.3 vs. 1.3) were recorded in group B vs. group A if responsive buffaloes are considered (n = 12) and the number of corpora lutea was highly correlated with the number of embryos (r = 0.65; P < 0.05). In conclusion, progesterone supplementation during the first 2 days of the superovulation treatment seems to enhance the recovery rate in buffalo species. A high ovulation rate, associated with a high number of corpora lutea, can represent a parameter for estimating embryo recovery.
No preview · Article · Aug 2010 · Tropical Animal Health and Production