Rossella Di Palo

Comparison of pregnancy rates with two estrus synchronization protocols in Italian Mediterranean Buffalo cows

The aim in this study was to compare two estrus synchronization protocols in buffaloes. Animals were divided into two groups: Group A (n=111) received 100 microg GnRH on Day 0, 375 microg PGF(2alpha) on Day 7 and 100 microg GnRH on Day 9 (Ovsynch); Group B (n=117) received an intravaginal drug release device (PRID) containing 1.55 g progesterone and a capsule with 10mg estradiol benzoate for 10 days and were treated with a luteolytic dose of PGF(2alpha) and 1000 IU PMSG at the time of PRID withdrawal. Animals were inseminated twice 18 and 42 h after the second injection of GnRH (Group A) and 60 and 84 h after PGF(2alpha) and PMSG injections (Group B). Progesterone (P(4)) concentrations in milk samples collected 12 and 2 days before treatments were used to determine cyclic and non-cyclic buffaloes, and milk P(4) concentrations 10 days after Artificial insemination (AI) were used as an index of a functional corpus luteum. Cows were palpated per rectum at 40 and 90 days after AI to determine pregnancies. All previously non-cyclic animals in Group B had elevated P(4) (>120 pg/ml milk whey) on Day 10 after AI. Accordingly, a greater (P<0.01) relative percentage of animals with elevated P(4) 10 days after AI were observed in Group B (93.2%) than in Group A (81.1%). However, there was no difference in overall pregnancy rates between the two estrus synchronization protocols (Group A, 36.0%; Group B 28.2%). When only animals with elevated P(4) on Day 10 after AI were considered, pregnancy rate was higher (P<0.05) for animals in Group A (44.4%) than Group B (30.3%). The findings indicated that treatment with PRID can induce ovulation in non-cyclic buffalo cows. However, synchronization of estrus with Ovsynch resulted in a higher pregnancy rate compared with synchronization with PRID, particularly in cyclic buffalo.

Bovine and buffalo in vitro embryo production using oocytes derived from abattoir ovaries or collected by transvaginal follicle aspiration.

This study was undertaken in order to evaluate the effect of oocyte source (live animals and abattoir ovaries) on subsequent embryo development in buffalo (Bubalus bubalis). Cow ovaries were also collected as oocyte donors for in vitro embryo production (IVEP). Three hundred thirty-eight oocytes were recovered by ovum pick up (OPU, Group A) from 8 pluriparous buffalo cows, while 1127 and 1457 oocytes were aspirated, respectively, from buffalo (Group B) and bovine (Group C) slaughterhouse ovaries. Cumulus enclosed oocytes (COCs) suitable for IVEP were in vitro matured (IVM), fertilized (IVF) and cultured (IVC) to the tight morula (Tm) and blastocyst (Bl) stage. Within buffalo species Group A had a higher Bl yield (29.7 % versus 19.9%; P<0.05) and a lower proportion of embryos arrested at Tm stage (11.1% versus 22.3%; P<0.05) than Group B. Within slaughterhouse groups cattle oocytes had a higher cleavage rate (83.9% versus 64.8%; P<0.05) and yielded 49.2% more blastocysts than buffalo. However, when data are related to the total number of cleaved oocytes, only 13.7% more blastocysts were produced in cattle than in buffalo.In conclusion, in buffalo species the source of oocytes significantly affected post-fertilization embryo development, as demonstrated by the higher Bl yields derived from OPU-derived oocytes.A higher overall IVEP efficiency, mainly related to the higher cleavage rate, was recorded in cattle compared with buffalo when ovaries from an abattoir were used as oocyte donors.

The effect of season on oocyte quality and developmental competence in Italian Mediterranean buffaloes (Bubalus bubalis).

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.

Sex determination of buffalo embryos ( Bubalus bubalis ) by polymerase chain reaction

The aim of this study was to identify a simple, rapid method for sex determination of in vitro produced buffalo embryos, amplifying Y-chromosome-specific repeat sequences by polymerase chain reaction (PCR). Buffalo oocytes collected from slaughtered animals were matured, fertilised and cultured in vitro for 7 days. On day 7 embryos were evaluated and divided in to six groups according to developmental stage (2, 4, 8, 16 cells, morulae and blastocyst). Each embryo was stored singly in phosphate-buffered saline at -20 degrees C until PCR. Two different methods of extraction of DNA were compared: a standard procedure (ST), using a normal extraction by phenol-chloroform, isoamyl alcohol and final precipitation in absolute ethanol and a direct procedure (DT), using a commercial kit (Qiaquik-Qiagen mini blood). A pair of bovine satellite primers and two pairs of different bovine Y-chromosome-specific primers (BRY4.a and BRY.1) were used in the PCR assay on embryos and on whole blood samples collected from male and female adult buffaloes, used as control. The trial was carried out on 359 embryos (193 for ST and 166 for DT). When DNA samples from blood were amplified, the sex determined by PCR always corresponded to the anatomical sex. Embryo sexing was not possible in two embryos in ST and one embryo in DT. Both extraction protocols recovered sufficient quantities of target DNA at all developmental stages, but the time required for the ST (24 h) limits its use in embryo sexing and supports the use of commercial extraction kits (5 h).

Genetic parameters for milk yield analyzed by test-day models in Murrah buffaloes in Brazil

New statistical models for genetic parameters estimation based on longitudinal data have been proposed. In this study, we considered the data of 47,614 test-day milk yields from 1,578 buffaloes, with 4,757 complete lactations, calving from 1985 to 2006. Single-, two- and multiple trait analyses were used and variance components were estimated by Restricted Maximum Likelihood. The model used to evaluate the milk yield trait at 305 days (MY305) included: i) herd-year-calving season (contemporary group) and milking number as fixed effects; ii) buffalo age at calving as covariate (linear and quadratic effect); iii) additive genetic, permanent environmental, residual and the animal as random effects. The same effects were included in the test-day milk yield model except the contemporary group, defined as herd-year-test day. The heritability estimations for TDMY vary from 0.13 to 0.23 for single-trait analyses, from 0.13 to 0.24 for two-trait analyses and from 0.15 to 0.24 for the multiple-trait analyses. The results obtained for each of the three models showed that the higher heritability estimations were always obtained in the third test month. The genetic correlations between the TDMY and MY305 were high and positive. In conclusion, the TDMY trait use into the selective process will promote genetic changes similar to the ones obtained through the MY305.

Microbiological quality of raw donkey milk from Campania Region

Microbiological quality of raw milk from eight healthy donkeys reared in Campania Region was investigated. A total of 152 samples were analyzed in order to evaluate the milk safety status trough monitoring mesophilic total bacterial count (TBC) at 32°C and 20°C, psychrophilic TBC at 5°C, Enterobacteriaceae, Salmonella spp., Listeria monocytogenes, Staphylococcus aureus and somatic cell count (SCC). The ranges for mesophilic bacteria at 32°C, 20°C and psy- chrophilic bacteria at 5°C were, respectively, 2.80–4.00 Log CFU/mL, 2.84–3.92 Log CFU/mL and 1.27–2.12 Log CFU/ml. Enterobacteriaceae showed a load ranging between 0.68–1.93 Log CFU/mL. No pathogenic bacteria were isolated. Estimated SCC values were always under 50.000 cells/mL. Additionally quantitative changes of bacterial population in raw bulk milk during eight storing days at 8°C and 3°C, were evaluated. Firstly, fresh bulk milk was contaminated by bacteria with a mean TBC at 32°C and 20°C of 2.71 Log CFU/mL and 2.64 Log CFU/mL, respectively, whereas TBC at 5°C and Enterobacteriaceae were not detected. After eight days of storage at 8°C, TBC at 32°C, 20°C and Enterobacteriaceae increased by three Log and TBC at 5°C by five Log. On the other side, after eight days of storage at 3°C no gradual Log increase was detected. Our results showed that donkey milk could be a good healthy ingredient for feeding where good hygienic procedures are applied and storage is kept at temperature lower than 3°C.

Long term effect of Ovum Pick-up in buffalo species.

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.

Sperm DNA assays and their relationship to sperm motility and morphology in bulls (Bos Taurus).

The relationship among sperm DNA assays in bulls with different sperm motility and morphology measures has not been reported. The objectives of the present study were to (1) describe Comet assay measures and examine their repeatability (inter- and intra-assay); (2) compare sperm DNA quality assays (i.e., Sperm Chromatin Structure Assay-SCSA; alkaline and neutral Comet assays and Sperm Bos Halomax assay-SBH) in two groups of bulls selected on either greater and lesser sperm motility and morphology (greater compared with lesser); (3) determine the relationship among DNA assays and sperm motility and morphology values. Inter-assay repeatability was greater for the neutral Comet assay as compared to the alkaline Comet assay. Intra-assay repeatability was greater than inter-assay repeatability for both Comet assays. Comet assay dimension measures and percentage tail DNA were the most repeatable for both Comet assays. Among sperm DNA quality assays, only SCSA measures and neutral Comet assay Ghosts (% Ghosts), head diameter and area, and comet area were different between greater and lesser sperm quality groups (P<0.05). The SCSA measures were inversely correlated with neutral Comet head measures (diameter, area, and intensity) and positively with percentage Ghosts (P<0.05). The % Ghosts and COMP-αt were correlated with some measures of sperm morphology and sperm motility. The neutral Comet assay was more appropriate for sperm evaluation than the alkaline Comet assay for distinguishing among groups with different sperm quality.

Milk protein and cheese yield in buffalo species

Abstract Buffalo milk samples differing significantly for cheese yield values were analysed by 2D electrophoresis in order to outline a protein profile, with specific regards to k-casein fractions. Four buffaloes, two of which showing high cheese yield and two with low cheese yield selected from a group of 135 subjects were chosen for the proteomic analyses. Six main spots in 2D gels were recognized as αs1-, αs2-, β- and k-casein, α-lactoalbumin, β-lactoglobulin. The main visible differences in the 2D gels between buffaloes with high vs. low cheese yield were found in the appearance of the four k-casein spots (spots numbers:20, 19, 16, 18) which differ in the number of phosphorilation and glycosilation. The area and the intensity of the four spots were calculated by using Melanie II (Bio-Rad) software. Samples with high cheese yield showed higher value of the by-products: area x intensity of spot 16, correspondent to k-casein with one phosphorilation site, and lower values of spots 19 and 20, of k-casein with more than one phosphorilation site and glycosilated.

Incidence of pregnancy failures in buffaloes with different rearing system

During a three year trial, 380 corpora lutea were found in lactating buffaloes and the ratios of not pregnant buffaloes/corpora lutea (NP/CL) were analysed by chi-square test between: years (1999 vs. 2000 vs. 2001), days open (<120 days vs. >120 days; DO1 and DO2 groups respectively), January–March vs. April–August calving periods, presence vs. absence of swimming-pool (G1 group vs. G2 group respectively). Data were analysed by chi-square test. NP/CL ratios increased throughout the years (7.53%A vs. 26.32%Bb vs. 41.73%Bc in 1999, 2000 and 2001 respectively) due to a progressive paddock overcrowding and were higher in DO2 groups and during the January-March period. The swimming pool presence was able to reduce significantly the NP/CL incidence (18.01%A vs. 33.14%B in G1 and G2 groups, respectively) with a significant effect only in the April-August period (18.62%A vs. 34.96%B in G1 and G2 respectively). NP/CL ratio, as expression of anomalous oestrous cycle and embryonic mortality, may be proposed as a specific tool for evaluating buffalo welfare.