Trypan blue/giemsa staining to assess sperm membrane integrity in salernitano stallions and its relationship to pregnancy rates.

Aim of this study was to test the reliability of Trypan blue/Giemsa staining to evaluate sperm membrane integrity, acrosomal intactness and morphology in stallion to verify whether it could be applied in vitro as useful tool for sperm fertilizing ability. Fertility data on inseminated mares were collected to evaluate the relationship of sperm quality to pregnancy rates. Forty-one ejaculates were collected from 3 stallions of Salernitano Horse Breed and evaluated for gross appearance, volume, visual motility and membrane integrity with Trypan blue/Giemsa staining and thirty-five mares were inseminated during the breeding season from April to July. Differences among stallions were found in volume, sperm concentration (p < 0.05) and visual motility (p < 0.01). A decrease in sperm motility, concentration (p < 0.05) and total sperm number was found in June-July (p < 0.01). Live sperm with intact acrosome (LSIA) and proximal droplets (PD) were lower (p < 0.01) in June-July, while acrosome reacted sperm (ARS) percentage increased (p < 0.05). No fertility differences were found among stallions with an average fertility per cycle of 44.6% and a pregnancy rate of 68.6%. Higher percentages of LSIA were found in the ejaculates used to inseminate mares that became pregnant vs those used in mares not pregnant (p < 0.05). The significance of LSIA as test variable to verify the reliability of Trypan blue/Giemsa staining was confirmed by Receiver operating characteristic ROC analysis and the sensitivity of the test was 85% at a cut-off value of 48% LSIA. Trypan blue-Giemsa showed to be an accurate method that can be applied on field to evaluate sperm membrane integrity and to identify poor-quality ejaculates.

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.

Efficacy of PGF(2α) on pre-ovulatory follicle and corpus luteum blood flow.

The aim of this study was to evaluate the effect of cloprostenol administration on the blood flow of pre-ovulatory follicle (PF) and corpus luteum (CL), progesterone secretion and pregnancy outcome in buffaloes subjected to AI. The trial was performed on 75 Italian buffaloes at 182 ± 8 days in milk. Synchronized animals were randomly divided into two groups on the day of oestrus: Group T (n = 37) received a 0.524 mg intramuscular injection of cloprostenol and Group C (n = 38) received saline. Ultrasound examinations of the ovaries were performed 5 h after AI on the PF and 10 and 20 days after AI on the CL. Resistive (RI) and pulsatily index (PI) were calculated by colour-Doppler mode in each examination. Blood samples were collected on days 10, 20 and 25 after AI for progesterone assay and 25 days after AI, ultrasonography was performed to assess pregnancy, which was confirmed on day 45. Subjects pregnant on day 25 but not on day 45 were considered to have undergone late embryonic mortality (LEM). Statistical analysis was performed by anova. No differences were found in PF dimensions, CL size and blood flow on day 10 and 20 after AI between treated and control groups. Pre-ovulatory follicle area was higher in buffaloes that resulted pregnant on day 25 after AI compared to those that were non-pregnant (2.13 vs 1.66 cm in pregnant and non-pregnant buffaloes, respectively), while non-pregnant buffaloes showed higher values of RI (0.49 vs 0.30; p < 0.05) and PI (1.0 vs 0.37; p = 0.07) compared to pregnant subjects. Treatment by cloprostenol did not influence pregnancy rate both on day 25 (31/75; 41.3%) and 45 (27/75; 36.0%), progesterone levels and incidence of LEM (4/31; 12.9%). In conclusion, cloprostenol administration at the time of AI does not seem to affect PF and CL blood flow.

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.

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.

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.

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.

Short communication: Detection of human Torque teno virus in the milk of water buffaloes (Bubalus bubalis).

Forty-four raw milk and 15 serum samples from 44 healthy water buffaloes reared in Caserta, southern Italy, the most important region in Europe for buffalo breeding, were examined to evaluate the presence of Torque teno viruses (TTV) using molecular tools. Furthermore, 8 pooled pasteurized milk samples (from dairy factories having excellent sanitary conditions) and 6 Mozzarella cheese samples were also tested. Four of the cheese samples were commercial Mozzarella cheese; the remaining 2 were prepared with TTV-containing milk. Human TTV were detected and confirmed by sequencing in 7 samples of milk (approximately 16%). No TTV were found in serum, pooled pasteurized milk, or Mozzarella cheese samples. The samples of Mozzarella cheese prepared with TTV-containing milk did not show any presence of TTV, which provides evidence that standard methodological procedures to prepare Mozzarella cheese seem to affect viral structure, making this food fit for human consumption. The 7 TTV species from water buffaloes were identified as genotypes corresponding to the tth31 (3 cases), sle 1981, sle 2031, and NLC030 (2 cases each) human isolates. Although cross-species infection may occur, detection of TTV DNA in milk but not in serum led us to believe that its presence could be due to human contamination rather than a true infection. Finally, the mode of transmission of TTV has not been determined. Contaminated of the food chain with TTV may be a potential risk for human health, representing one of the multiple routes of infection.

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.

Epizootic abortion related to infections by Chlamydophila abortus and Chlamydophila pecorum in water buffalo (Bubalus bubalis).

Water buffalo (Bubalus bubalis) are affected by high rates of embryonic mortality and abortion related to infectious diseases and non-infectious factors. A number of viral and bacterial infections have been associated with reproductive failure, but there is limited information on the role of chlamydial infections. In order to investigate the presence and the role of Chlamydiaceae in water buffalo a retrospective study was performed in a herd with a history of reproductive failure. During an 11-month period, the pregnant heifers suffered an abortion rate of 36.8% between the 3rd and 7th month of pregnancy. Antibodies to Chlamydiaceae were detected in 57% of the aborted cows, and in 0% of the overtly healthy cows used as control. By a nested-PCR assay, three of 14 vaginal swabs from aborted animals tested positive for Chlamydophila agents and, additionally, three out of seven aborted fetuses tested positive for Chlamydophila spp., with two being co-infections by Cp. abortus and Cp. pecorum and one being characterised as Cp. abortus. Sequence analysis of the amplicons confirmed the results of the nested-PCR. The presence of anti-Chlamydiaceae antibodies in more than half of the aborting animals (P<0.002) and the detection of Chlamydophila agents in several fetal organs and in the vaginal swabs are consistent with the history of abortions observed in the herd and suggest an abortifacient role by Chlamydophila spp. in water buffalo (B. Bubalis) herds.

Corpus luteum function and embryonic mortality in buffaloes treated with a GnRH agonist, hCG and progesterone.

The effect of treatment with a GnRH agonist, hCG or progesterone (P(4)) on corpus luteum function and embryonic mortality was investigated in buffaloes inseminated during mid-winter. Italian Mediterranean buffaloes (n=309) were synchronized using the Ovsynch with timed-AI program and mated by AI at 16 h (Day 0) and 40 h after the second injection of GnRH. On Day 5, buffaloes were randomly assigned to four groups: Control (no treatment, n=69), GnRH agonist (buserelin acetate, 12.6 microg, n=73), hCG (1500 IU, n=75) and P(4) (PRID without E(2) for 10 days, n=77). Progesterone (pg/ml) was determined in milk whey on Days 5, 10, 15 and 20 and pregnancy diagnosis was undertaken on Day 26 by ultrasound and Day 40 by rectal palpation. Treatment with buserelin and hCG increased (p<0.05) P(4) on Day 15 compared with controls (456+/-27, 451+/-24 and 346+/-28 pg/ml, respectively). Buffaloes treated with a PRID had intermediate P(4) concentrations (380+/-23 pg/ml). Embryonic mortality between Days 26 and 40 (22.9%) and pregnancies at Day 40 (48.9%) did not differ between treatments. A higher (p<0.01) P(4) concentration was found on Day 20 in pregnant animals compared with non-pregnant and embryonic mortality buffaloes, which did not differ. In summary, buserelin and hCG increased P(4) concentrations on Day 15 but this was not associated with a reduced incidence of embryonic mortality in buffaloes during mid-winter.

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.

Effects of using vasectomized bulls in artificial insemination practice on the reproductive efficiency of Italian buffalo cows.

The effects of the presence or absence of vasectomized male buffaloes on the reproductive efficiency of buffalo cows (n = 396) undergoing artificial insemination (AI) was studied on six farms owned and operated by a single consortium. Lactating animals were separated into two groups of various sizes on each farm and kept under semi-range conditions. Vasectomized bulls were present in one group at a bull/empty-cow ratio of 1:30. No bulls were present in the other group. Reproductive efficiency between the two groups over a period of 3.5 months was compared and evaluated on the basis of: 1) the number of spontaneous overt estruses associated with either feeble or intense signs of estrous behaviour; 2) the number of functional estrous cycles, i.e. estrous cycles with luteal phases defined as normal, based on specified progesterone concentrations in milk or blood plasma 8-10 days after estrus; 3) the number of consecutive functional estrous cycles in cases of induced estrus; and 4) pregnancy rate. Groups with bulls present demonstrated a significantly higher reproductive efficiency than groups without them. There was a higher incidence of spontaneous estrus (92 versus 69%; P < 0.01); spontaneous estrus of high intensity (62.2 versus 31.1%; P < 0.01); and higher incidence of functional estrous cycles following both spontaneous (65.8 versus 57.1%) and induced (77.0 versus 59.5%; P < 0.05) estrus. Exposure to vasectomised bulls also increased the incidence of consecutive functional estrous cycles (90.5 versus 68.1%; P < 0.01), and the pregnancy rate in cows inseminated at spontaneous (42.5 versus 18.9%; P < 0.01) or induced (51.1 versus 33.3%; P < 0.05) estrus. Overall pregnancy rate did not differ significantly between cows inseminated at induced or spontaneous estrus, although in the absence of bulls, pregnancy rate per AI was higher in cows inseminated at induced than at spontaneous estrus (33.3 versus 18.9%).