R. Di Palo

Efficacy of PGF2α 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.

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.

Ca and P in buffalo milk: curd yield and milk clotting parameters

Abstract Aim of this study was to evaluate the mineral milk content and its relationship with the cheese yield and the rennet coagulation properties. Ca and P content, total and soluble, was determined on 70 milk samples along with the physic-chemical composition, cheese yield and coagulation parameters. Total Ca and P contents were 170,57±14,41mg·dl-1 and 145,34±26,87 mg·dl-1,with a Ca/P ratio of about 1.2. Fresh cheese yield was on average 261.7 ± 25.4 gr·l-1 of milk and was influenced by both milk quality and Ca and P contents (R2=0.82). The average rennet coagulation parameters had the following values: R = 14,20±3,82; K20= 1,73±0,97 and A30= 46,01±8,81. R values was influenced positively (R2=0.68) by milk pH, protein and fat content and negatively by the Ca/P ratio while shorter K20 value were linked to low micellar Ca and higher soluble P (R2=0.46). The A30 was negatively influenced by milk pH, fat/protein ratio and positively by soluble Ca and P content and micellar P % (R2 =0.50).

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.

Evaluation of buffalo semen by Trypan blue/Giemsa staining and related fertility in vitro

Abstract The aim of this work was to verify the feasibility of an easy, quick double staining technique for evaluation of frozen-thawed semen to predict the fertilizing capability in vitro of buffalo bulls. In Experiment 1, frozen-thawed semen from 6 bulls was stained with double Trypan blue/ Giemsa and the incidence of acrosome-intact live (AIL), acrosome-intact dead (AID), acrosome-lost live (ALL) and acrosome-lost dead (ALD) sperm was recorded. In Experiment 2, sperm from the same bulls were used to fertilize in vitro matured oocytes. The data obtained confirm that there is a strong “bull effect” in buffalo species, with differences in the percentage of AIL sperm at thawing, in cleavage and blastocyst rates among bulls. Interestingly, it was found that this staining technique can be used for a preliminary screening to select semen to use for IVF, as shown by the correlation existent between the percentages of acrosome-intact viable sperm cells at thawing and the blastocyst yields for 4/6 bulls.

Relationship between lactodinamographic and characteristics of buffalo milk

Abstract The aim of this study was to evaluate the relationships between 44 components and/or characteristics of milk samples collected every 50 days from 60 buffaloes (326 samples and 14,344 values). The animals were half sib (same father or same mother) and were bred in two farms. Significant associations but with a low values of coefficient of correlation were present, demonstrating that lactodinamographic parameters marginally affect cheese yield.

Strategies to reduce embryonic mortality in buffalo cows

Abstract The aim of the present study was to examine whether treatment with a GnRH agonist, hCG or P4 on Day 25 after AI increased P4 concentrations and reduced the incidence of embryonic mortality (EM) in pregnant buffaloes mated in mid-winter in a Mediterranean environment. The trial was carried out in two farms characterized, in previous years, by low (LEM Group), 153 buffaloes (DIM=150±7 days), and high (HEM Group), 284 buffaloes (DIM=163±5 days), incidence of embryo mortality. Animals were synchronized by Ovsynch-TAI Program and artificially inseminated. On day 25, pregnant buffaloes were randomly assigned to four groups: Control (no treatment), GnRH agonist (buserelin acetate, 12.6 µg), hCG (1500 IU) and P4 (341 mg of P4 i.m. every 4 days for three times). Progesterone (pg/ml) was determined in milk whey on Days 10, 20 and 25 after AI in all buffaloes and in Days 30 and 45 only in buffaloes pregnant on day 25 and assigned to four groups of treatment. Pregnancy diagnosis was undertaken on Day 45 by ultrasound. All treatments increased P4 milk whey and reduced embryonic mortality in buffalo cows bred in the farm characterized by high EM.

Mediterranean river buffalo oxytocin-neurophysin I (OXT) gene: structure, promoter analysis and allele detection

Abstract Oxytocin (OXT) is a very abundant nonapeptide neurohypophysial hormone implicated in several aspects of reproduction, including social, sexual and maternal behaviour, induction of labour and milk ejection. The nucleotide sequence of the whole OXT-neurophysin I encoding gene (OXT) in Mediterranean river buffalo was determined, plus 993 nucleotides at the 5’ flanking region. Buffalo oxytocin gene sequence analysis showed two transitions in the promoter region (C→T in position – 966 and G→A in position – 790) and one transversion G→T at the 170th nucleotide of the second exon, responsible for the Arg97→Leu aa substitution which identifies an allele named OXT B. A PCR-RFLP based method for a rapid identification of carriers of these alleles has been developed.

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.

Influence of temperature and time during ovary transportation on in vitro embryo production efficiency in the buffalo species (Bubalus bubalis)

Abstract The aim of this study was to evaluate the influence of temperature during ovary collection/transportation and that of the time interval between ovary collection and processing in the laboratory, on in vitro embryo production efficiency in buffalo species. A retrospective analysis of data collected over the last 4 years in our lab was carried out on 3461 oocytes, recovered over 120 replicates. No differences in oocytes developmental competence were observed in relation to the time interval between ovary collection and processing in the laboratory (3-6 h). On the contrary, a correlation was found between oocyte developmental competence, evaluated in terms of cleavage and blastocyst rate, and temperature during ovary collection/transportation. In particular, lowering temperature during ovary transportation significantly improved both cleavage (74.6 % vs 65.8 and 63.1 % for temperature ranges of 25-30°C, 30-34°C and 34-37°C, respectively ; P<0.05) and blastocyst rates (30.9 % vs 21.8 and 22.7 % for temperature ranges of 25-30°C, 30-34°C and 34-37°C, respectively ; P<0.05). It was concluded that extending the time interval between ovary collection and processing to 6 h is not detrimental for buffalo oocyte developmental competence. Furthermore, lowering temperature during ovary collection and transportation increases the in vitro embryo production efficiency in buffalo species.