Effect of resveratrol supplementation during culture on the quality and cryotolerance of bovine in vitro produced embryos.

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), 0.25µM (n=422), 0.5µM (n=447) and 1µM resveratrol (n=416). On Day 7 (IVF=Day 0) blastocysts were vitrified by cryotop in 16.5% ethylene glycol, 16.5% dimethyl sulfoxide and 0.5M 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 48h culture. Blastocysts cultured with (0.5µM) 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 1µM resveratrol). However, 0.5µM 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 48h post-warming culture. Blastocysts produced in the control and in 0.5µM 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.

Influence of oocyte donor on in vitro embryo production in buffalo.

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.

Influence of season on corpus luteum structure and function and AI outcome in the Italian Mediterranean buffalo (Bubalus bubalis).

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.

The influence of gamete co-incubation length on the in vitro fertility and sex ratio of bovine bulls with different penetration speed.

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.

Pregnancy rates following AI with sexed semen in Mediterranean Italian buffalo heifers (Bubalus bubalis).

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.

Genetic parameter estimates for buffalo milk yield, milk quality and mozzarella production and Bayesian inference analysis of their relationships.

Buffalo milk has excellent physical and chemical qualities as a consequence of the high percentage of constituents. This milk property is desirable for the dairy industry because it facilitates manufacture of mozzarella cheese. We estimated genetic parameters for milk yield, milk fat and protein and their effects on mozzarella cheese production using Bayesian inference. Using information from 4907 lactation records of buffaloes, genetic and non-genetic parameters were estimated for accumulated 305-day milk yield (MY), milk fat (%F) and protein (%P) percentages and mozzarella production per lactation (MP). The (co)variance components were obtained by Bayesian inference using a multiple trait model, which included as fixed effects contemporary group, milking number and buffalo age at calving as covariables (linear and quadratic), along with the additive genetic, permanent environmental and residual random effects. Mean a posteriori heritability distributions for MY, %F, %P, and MP were 0.25, 0.30, 0.38, and 0.23, respectively. The genetic correlation estimates between MY with %P and %F were negative and moderate. Positive genetic correlation estimates varying from 0.19 (%P/MP) to 0.95 (MY/MP) were obtained among the traits. Milk yield, milk components, and mozzarella production in Murrah buffaloes have enough genetic variation for selection purposes. We conclude that selection to increase milk yield would be effective in improving mozzarella production.

Enhancing reproductive performance in domestic dairy water buffalo (Bubalus bubalis).

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.

Ion, protein, phospholipid and energy substrate content of oviduct fluid during the oestrous cycle of buffalo (Bubalus bubalis).

The aim of this research was to analyse the composition of oviduct fluid (ODF) in buffalo cows at different oestrous cycle phases to fulfil the requirements of buffalo embryos in vitro. ODF was collected by chronic cannulation from three cows that were synchronized by administering a synthetic prostaglandin. Based on hormonal profiles, the pre-ovulatory, ovulatory, post-ovulatory and luteal phases of the oestrous cycle were defined. The volume of ODF produced (ml/24 h) was influenced by the oestrous cycle, with values (mean ± SE) around ovulation (1.0 ± 0.2) greater (p < 0.05) than in both the luteal (0.4 ± 0.1) and the post-ovulatory phases (0.5 ± 0.1), but not different from the intermediate values in the pre-ovulatory phase (0.8 ± 0.2). Among cycle phases, no differences were found in sodium, potassium, calcium and magnesium concentrations (130.0 ± 1.1, 5.1 ± 0.3, 2.8 ± 0.1 and 0.59 ± 0.04 mmol/l respectively). Interestingly, the chloride secretion (µm/24 h) was higher (p < 0.05) at ovulation (150.2 ± 16.5) than during both the luteal (73.7 ± 22.0) and the post-ovulatory phases (63.7 ± 11.2), with intermediate values in the pre-ovulatory phase (113.4 ± 23.5). Glucose concentration (mmol/l) was higher (p = 0.056) in the pre-ovulatory phase (0.06 ± 0.02) than in the luteal (0.02 ± 0.01) and post-ovulatory (0.02 ± 0.01) phases but not different from values in the ovulatory phase (0.04 ± 0.02). Concentrations of pyruvate and lactate among oestrous cycle phases were similar (0.08 ± 0.01 and 1.0 ± 0.1 mmol/l respectively). The total quantity of phospholipids (µmol/24 h) was greater (p < 0.05) at ovulation (0.21 ± 0.02) compared with the luteal, pre-ovulatory and post-ovulatory phases of the cycle (0.09 ± 0.02, 0.13 ± 0.02 and 0.09 ± 0.01 respectively). No differences were found in either the protein concentration (1.8 ± 0.3 mg/ml) or the quantity of proteins secreted in 24 h (1.8 ± 0.4 mg) among oestrous cycle phases. In conclusion, this study provides the first characterization of buffalo ODF during the oestrous cycle, showing species-specific differences that may be useful for developing suitable media for buffalo in vitro embryo production.

Cryotop vitrification of buffalo (Bubalus bubalis) in vitro matured oocytes: effects of cryoprotectant concentrations and warming procedures.

The aim of the work was to evaluate the in vitro developmental competence of in vitro-matured buffalo oocytes after Cryotop vitrification (CTV) and in vitro fertilization (IVF). To optimize parameters, two cryoprotectant (CP) concentrations and two warming-dilution procedures were applied. Oocytes were vitrified in 16.5% ethylene glycol (EG), 16.5% dimethylsulphoxide (DMSO) and 0.5 M sucrose in Groups A and C, and in higher CP concentrations (20% EG, 20% DMSO and 0.5 M sucrose) in Groups B and D. Warming was performed in 1.25 M sucrose for 1 min, then in 0.62, 0.42 and 0.31 M sucrose, 30 s each (Groups A and B), or in 0.25 M sucrose for 1 min and in 0.15 M sucrose for 5 min (Groups C and D). After warming, the oocytes were fertilized and cultured in vitro. Survival rate post-warming was lower in Group D (83.6%) than in Groups A and B (92.4 and 92.8%, respectively), while intermediate values were found in Group C (85.7%). Survival rates at 24 h decreased in Groups C and D (52.0% and 50%, respectively) and remained high in Groups A and B (84.0% and 85.6%, respectively), thus indicating that the dilution of CP after warming is critical for buffalo oocyte cryopreservation. Similar differences were also observed in cleavage rates (42.7%, 55.3%, 28.4% and 36.3% for Groups A, B, C and D, respectively) whereas no differences in blastocyst rates were found among groups (6.4%, 7.8%, 5.9% and 6.9% for Groups A, B, C and D, respectively). Blastocyst production after IVF of vitrified oocytes proves the feasibility of CTV in buffalo species.

Corpus luteum function and pregnancy outcome in buffaloes during the transition period from breeding to non-breeding season.

The aim in this study was to investigate corpus luteum function and embryonic loss in buffaloes mated by artificial inseminations (AI) during the transitional period from breeding to non-breeding season. The study was carried out using 288 multiparous Italian Mediterranean Buffalo cows at 110 ± 4 days in milk. The buffaloes were mated by AI after synchronization of ovulation by the Ovsynch-TAI protocol 25 days after AI buffaloes underwent trans-rectal ultrasonography to assess embryonic development. Pregnancy diagnosis was confirmed on Days 45 and 70 after AI by rectal palpation. Buffaloes pregnant on Day 25 but not on Day 45 were considered to have undergone late embryonic mortality (LEM), whilst buffaloes pregnant on Day 45 but not on Day 70 were considered to have undergone foetal mortality (FM). Corpus luteum size and blood flow were determined by real-time B-mode/colour-Doppler on day 10 after AI in 122 buffaloes. The resistive index (RI) and pulsatility index (PI) were recorded at the time. Milk samples were collected on Days 10, 20 and 25 after AI in all inseminated buffaloes for the assay of whey P4 concentrations. Data were analysed by anova. Pregnancy rate on Day 25 after AI was 48.6% (140/288) and declined to 35.4% (102/288) and 30.6% (88/288) by Day 45 and Day 70 respectively. The incidences of LEM and FM were respectively 27.1% (38/140) and 13.7% (14/102). Pregnant buffaloes had greater (p < 0.01) whey concentrations of P4 from Day 20 onwards than buffaloes which showed LEM, whilst P4 in buffaloes that showed FM did not differ from the other two groups on Day 10 and Day 20. Corpus luteum blood flow on Day 10 after AI showed higher RI (p < 0.05) and PI (p = 0.07) values in buffaloes that subsequently were not pregnant on Day 25 compared with pregnant buffaloes. Buffaloes that were not pregnant on Day 45 also had a higher (p = 0.02) RI value on Day 10 than pregnant buffaloes, whilst PI values on Day 10 did not differ for the two groups of buffaloes. It was concluded that blood flow to the corpus luteum on Day 10 after AI influences corpus luteum function as judged by P4 secretion and also embryonic development and attachment in buffaloes.

Is a delayed treatment with GnRH, HCG or progesterone beneficial for reducing embryonic mortality in buffaloes?

The aims of this study were to verify the efficacy of delayed hormonal treatments performed on day 25 post-insemination on pregnancy rate at 45 and 70 days in buffalo. The trial was performed on 385 buffaloes synchronized by the Ovsynch/TAI protocol and submitted to artificial insemination (AI). Twenty-five days after AI, pregnant animals were assigned to four treatments: (1) GnRH agonist (n = 52), 12 microg of buserelin acetate; (2) hCG (n = 51), 1500 IU of human chorionic gonadotrophin; (3) Progesterone (n = 47), 341 mg of P4 intramuscular (im) every 4 days for three times; (4) Control (n = 54), treatment with physiological saline (0.9% NaCl). Milk samples were collected on days 10, 20 and 25 after AI in all buffaloes to determine progesterone concentration in whey by radioimmunoassay method. Statistical analysis was performed by anova. Pregnancy rate on day 25 after AI was 52.9%, but declined to 41.8% by day 45, indicating an embryonic mortality (EM) of 21%. If only control group is considered, the incidence of EM was 38.9%. Pregnant buffaloes had higher (p < 0.01) progesterone concentrations on day 20 and 25 after AI than both non-pregnant buffaloes and buffaloes that showed EM. The treatments on day 25 increased (p < 0.01) pregnancy rate, although in buffaloes with a low whey progesterone concentration on day 20 and 25 after AI (n = 22); all treatments were ineffective to reduce EM.

Effect of osteopontin (OPN) on in vitro embryo development in cattle.

Fertility-related phosphoprotein osteopontin (OPN) is present in the bovine oviduct epithelium and fluid. The objectives were to determine the effects of OPN on percentages of cleavage and embryo development in vitro in cattle, and to assess the ability of OPN to induce in vitro capacitation of bovine sperm. In vitro-matured bovine oocytes were fertilized in the presence of 0, 10, 20, or 40 microg/mL OPN. There were greater percentages (P<0.01) of cleavage and compact morulae-blastocysts (79.7 and 43.3%, respectively) with 10 microg/mL OPN than in the control group (without OPN; 71.2 and 32.1%, respectively). Furthermore, percentages of advanced blastocysts were greater in the group receiving 40 microg/mL OPN versus control (56.4% vs. 42.0%, P<0.05). Capacitation was assessed by the ability of sperm to undergo the acrosome reaction after incubation with lysophosphatidylcholine. Semen from three bulls was incubated for 2h in either TALP medium alone (control) or with TALP medium containing 0.01 mM heparin, or with TALP medium containing 10 or 20 microg/mL OPN. Incubation with 10 and 20 microg/mL OPN produced more (P<0.01) capacitated sperm (14.4 and 13.6%, respectively) than the untreated control group (8.3%), but both untreated sperm and those treated with OPN had significantly fewer capacitated sperm than those treated with 0.01 mM of heparin (30.5%). In conclusion, OPN improved the efficiency of bovine in vitro embryo production and influenced sperm capacitation.

Effect of recombinant bovine somatotropin (bST) on follicular population and on in vitro buffalo embryo production.

The objective of this study was to evaluate the effect of bovine somatotropin (bST) on ovarian follicular population in buffalo heifers and its influence on oocyte quality, recovery rates and in vitro embryo production. We tested the hypothesis that bST treatment in buffalo females submitted to an ovum pick-up (OPU) program would improve the number of follicles recruited, oocyte quality and in vitro embryo production. A total of 10 heifers were assigned into two treatment groups: group bST (n=5; receiving 500 mg of bST in regular intervals) and control group (n=5; without additional treatment). Both groups were subjected to OPU sessions twice a week (every 3 or 4 days), for a total of 10 sessions per female, although due to procedural problems, only the first five OPU sessions produced embryos. The number of follicles and the diameters were recorded at all OPU sessions. The harvested oocytes were counted and classified according to their quality as either A, B, C, D or E, with A and B considered good quality. Cleavage and blastocyst production rates were evaluated 2 and 7 days after in vitro fertilization, respectively. The bST treatment increased the total number of antral follicles (>3mm in diameter; 12.2 compared with 8.7; p<0.05) and of small antral follicles (<5mm; 9.1 compared with 6.5; p<0.05) per OPU session. The bST also tended to increase the number of oocytes recovered per session (5.2 compared with 4.1; p=0.07), and enhanced the percentage of good quality oocytes (48.8% compared with 40.6%; p=0.07). bST showed no effect on cleavage and blastocyst production rates (p>0.05). The significant effects of performing repeated OPU sessions were decreasing the follicular population (p<0.001) as well as the number of follicles aspirated (p<0.001), and oocytes recovered (p<0.02). In conclusion, bST treatment improves the follicular population, demonstrating its possible application in buffalo donors submitted to OPU programs.

Delayed treatment with GnRH agonist, hCG and progesterone and reduced embryonic mortality in buffaloes.

The present study examined the effect of delayed treatment with tropic hormones and progesterone (P4) on embryonic mortality in buffaloes. Buffaloes with a conceptus on Day 25 after AI were assigned to the following treatments: Control (n=41), i.m. physiological saline; GnRH agonist (n=36), i.m. 12 microg buserelin acetate; hCG (n=33), i.m. 1500 IU hCG; P4 (n=38), i.m. 341 mg P4 every 4 days on three occasions. Control buffaloes had an embryonic mortality of 41.4% (17/41) between Days 25 and 45, and this was reduced (P<0.01) by treatment with GnRH agonist (11.1%, 4/36), hCG (9.0%, 3/33) and P4 (13.1%, 5/38). On Day 45, buffaloes treated with hCG and which ovulated had greater (P<0.05) concentrations of P(4) in whey (453 +/- 41 pg/ml) than buffaloes in the same treatment that did not ovulate (297 +/- 32 pg/ml). A similar but non-significant trend was observed for buffaloes treated with GnRH agonist. It was concluded from the findings that the treatment of buffaloes on Day 25 after AI with tropic hormones or P4 is beneficial to processes associated with embryonic implantation.

Sex chromosome abnormalities and sterility in river buffalo.

Thirteen male river buffaloes, 119 females with reproductive problems (which had reached reproductive age but had failed to become pregnant in the presence of bulls) and two male co-twins underwent both clinical and cytogenetic investigation. Clinical analyses performed by veterinary practitioners revealed normal body conformation and external genitalia for most females. However, some subjects showed some slight male traits such as large base horn circumference, prominent withers and tight pelvis. Rectal palpation revealed damage to internal sex adducts varying between atrophy of Mullerian ducts to complete lack of internal sex adducts (with closed vagina). All bulls had normal karyotypes at high resolution banding, while 25 animals (23 females and 2 male co-twins) (20.7%) with reproductive problems were found to carry the following sex chromosome abnormalities: X monosomy (2 females); X trisomy (1 female); sex reversal syndrome (2 females); and free-martinism (18 females and 2 males). All female carriers were sterile.

Effect of prostaglandin F2alpha at the time of AI on progesterone levels and pregnancy rate in synchronized Italian Mediterranean buffaloes.

The aims of this study were to evaluate the effects of an intravenous or intramuscular PGF2alpha analogue administration on the day of estrus on progesterone concentration and pregnancy rate in buffaloes undergoing artificial insemination (AI). To this end, two experiments were carried out. The first study was performed on 72 Mediterranean buffaloes synchronized by the Ovsynch-TAI Program. On the day of estrus only animals considered in heat were divided into four groups: Groups IVC and IMC received, respectively, an intravenous or intramuscular injection of cloprostenol (0.524 mg), whereas control Groups IVS and IMS received the same injections of saline. Milk samples were collected daily from each animal to assess progesterone concentration in the whey by RIA method. In addition on alternate days, buffaloes underwent transrectal ultrasound analysis. The second study was carried out on 385 buffaloes synchronized by the Ovsynch-TAI Program. On the day of AI, animals were divided in four groups, as described in experiment 1. Pregnancy rate was evaluated either on day 26 or day 45 and embryonic mortality rate was recorded. Statistical analysis was performed by ANOVA and chi2 test. A higher (P<0.05) progesterone concentration was recorded on day 11 (Day 0=estrus day) in Groups IVC and IMC compared to Groups IVS and IMS (351.6+/-129.7 and 355.8+/-112.2 pg ml(-1) vs. 239.8+/-81.1 and 243.6+/-90.5 pg ml(-1), respectively). Furthermore, a larger CL was recorded on the same day in treated vs. control groups (1.25+/-0.15 and 1.27+/-0.17 cm, respectively, in Groups IVC and IMC vs. 1.08+/-0.14 and 1.05+/-0.13 cm in IVS and IMS). In the second study, a higher pregnancy rate was observed in treated (IVC+IMC) vs. control (IVS+IMS) groups (46.7% vs. 30.7%; P<0.01), while no differences were recorded between treated groups. From these data, it can be concluded that either intravenous or intramuscular administration of PGF2alpha at the time of AI can enhance progesterone levels and pregnancy rate in buffaloes.

Importation of in vitro-produced Bubalus bubalis embryos from Italy into the United States: a case report.

On December 19, 2005, 14 in vitro-fertilized water buffalo (Bubalus bubalis) embryos, which had been cryopreserved by vitrification, were thawed and transferred into B. bubalis recipients in California. The embryos had been produced in Italy, following transvaginal oocyte pickup (TVOPU), with subsequent in vitro maturation, insemination, and culture. This case study relates our experience in meeting the regulatory criteria, established by the Animal Import/Export Office of the USDA-Animal and Plant Health Inspection Service (USDA-APHIS), in order to successfully import these embryos into the USA.

Freemartinism in river buffalo: clinical and cytogenetic observations.

During the last five years forty phenotypic female river buffalos with reproductive problems and two phenotypic male co-twins of females, raised in the provinces of Caserta and Salerno (southern Italy) underwent cytogenetic investigation. Of the 42 animals studied, 10 freemartins (8 females and 2 males) were found with variable percentages of male and female blood cells, the majority however showing similar percentages of both. Of the eight females, six showed normal body conformation, vagina and clitoris, while two showed some male traits (tight pelvis). The two males were apparently normal with only a reduced size of one testicle in one animal. Clinical observations performed in the internal reproductive organs of the female carriers by both rectal palpation (5 females) and direct observation after mating (3 females) revealed serious damages varying from complete lack of internal sex adducts (closed vagina) to hypoplasia of Mullerian ducts and absence (or atrophy) of ovaries. All freemartin females were sterile. To our knowledge, this is the first detailed description of freemartinism in river buffalo by combining clinical and cytogenetic analyses.