Characterization of leptin receptor gene in Bubalus bubalis and association analysis with body measurement traits.

Leptin has a pleiotropic effect on regulating appetite, energy metabolism, growth, reproduction, body composition and immunity. This property supports leptin and its receptor as candidate genes for evaluating genetic polymorphisms to associate with growth, milk yield and other economic traits. The aim of this study is to characterize the leptin receptor gene in Bubalus bubalis, to identify single-nucleotide polymorphism (SNP) sites in different coding and non-coding regions and to analyse potential associations between SNPs identified and the body measurements traits of growing buffalo heifers. A group of 64 animals were genotyped by direct sequencing and twenty-eight SNPs were detected. A sequence analysis revealed the presence of nine interesting SNPs in gene sequence. The association analysis of polymorphisms with the body measurements traits of growing buffalo heifers shows significant statistical effects on chest depth and sacrum height. Therefore according to the results obtained from this study, the leptin receptor gene appears to have potential effects on the body measurement traits of Bubalus bubalis.

Seasonality of plasma testosterone in males of four Mediterranean goat breeds and in three different climatic conditions.

The aims of the present work were to evaluate if a seasonality of plasma testosterone concentrations exists in Mediterranean male goats and it may be affected by climatic conditions. Testosterone concentrations were determined throughout 1 year in blood samples collected weekly from 24 males of 4 Mediterranean goat breeds (Ionica, Garganica, Maltese and Red Syrian), fed a constant diet, maintained without interactions with does and reared in 3 farms with different climates, in Italy. Plasma testosterone concentrations were affected by season (P<0.0001), being higher during summer (8.33+/-0.35 ng/mL) than during autumn (6.81+/-0.26 ng/mL), which were in turn higher than in winter (1.55+/-0.08 ng/mL) and spring (2.04+/-0.15 ng/mL). An effect of breed was recorded (P<0.001), Ionica bucks having the highest plasma testosterone means, Red Syrian and Maltese the lowest. The onset of the reproductive season was similar among the breeds. Ionica bucks had a later offset than Red Syrian and Maltese and a longer reproductive season than Maltese bucks (P<0.05). No effect of farm nor interaction breed x farm was recorded. Single sporadic peaks of plasma testosterone were recorded in many bucks, outside the main reproductive season, especially during spring. In conclusion the bucks of all the four breeds displayed a clear seasonality of plasma testosterone, with very low levels from January to May and high levels from July to November; hormone levels and pattern of seasonality were affected by breed and not by climate.

Postpartum ovarian follicular dynamics in primiparous and pluriparous Mediterranean Italian buffaloes (Bubalus bubalis).

The objective of this study was to monitor ovarian function in postpartum primiparous and pluriparous Mediterranean Italian buffaloes (Bubalus bubalis) during months of increasing daylength. Ovarian ultrasound monitoring was carried out for a total of 60 days from calving in 10 primiparous and 10 pluriparous buffaloes. Progesterone was determined from calving until a week after first postpartum ovulation. The study was undertaken during months of increasing day length. Time required for complete postpartum uterine involution was 31 +/- 1.0 and 33 +/- 1.3 days in primiparous and pluriparous buffaloes respectively (P = 0.1). The first postpartum ovulation was recorded on 4 primiparous and 8 pluriparous buffaloes (P = 0.16). Time for first postpartum ovulation to occur was 25.5 +/- 6.9 and 15.5 +/- 1.3 days in primiparous and pluriparous buffaloes, respectively (P = 0.07). Overall, 8 of the 12 first postpartum ovulations (66.6%) occurred in the ovary contra-lateral to the one bearing the gravidic CL, one out of 4 in primiparous and 3 out of 8 in pluriparous buffaloes (P = 1.0). Following a first postpartum ovulation, 3 primiparous and 4 pluriparous buffaloes displayed a complete wave of follicular development leading to a new ovulation. Ovulation following parturition was not recorded in 6 primiparous and two pluriparous buffaloes for the 60 days of ultrasound monitoring. Growth rate (mm/d) and largest size (mm) of first postpartum ovulating follicle was 0.95 +/- 0.18 and 1.07 +/- 0.07 (P = 0.4), and 13.5 +/- 0.8 and 14.1 +/- 0.4 (P = 0.4) in primiparous and pluriparous buffaloes, respectively. Following calving, the total number of available antral follicles (> or =2 mm) declined gradually towards the end of the study period. Follicles greater or equal to 3 mm in diameter on the contrary showed a prominent increase in the first 2 weeks from calving. The number of follicles greater or equal to 3 mm in diameter was significantly higher in the ovary contra-lateral to the one bearing the gravidic CL. A balance in the number of such follicles was reached toward the end of the first month. In conclusion, although some follicular activity was recorded in the ovaries of all buffaloes, true postpartum resumption of cyclicity in the months of increasing daylight hours was delayed in the majority of animals.

Hormonal stimulation and oocyte maturational competence in prepuberal Mediterranean Italian buffaloes (Bubalus bubalis).

The objective of this study was to determine the best combined hormonal treatment to utilize in order to obtain a high number of good quality in vivo and in vitro matured oocytes from prepuberal Mediterranean Italian buffalo calves (Bubalus bubalis). Transvaginal ultrasound follicular aspiration was employed to recover oocytes from antral follicles. Fifteen barn housed buffalo calves, between 5 and 9 months of age were used in this study and randomly divided into control (Group A) and treated groups. A commercially available preparation of 2000 IU eCG was administered to animals in the treatment groups, followed by 2000 IU of hCG given either 12 h (Group B), or 24 h (Group C) before ovum pick up (OPU). From the time of administration of eCG treatments, the best timing for hCG administration before OPU was determined and integrated with the administration of 500 IU of FSH-LH in a decreasing dosage protocol over 4 days (Group D). Expanded cumulus oocyte complexes (COCs) recovered from all groups were immediately fixed for later aceto-orcein staining. All other COCs were processed for in vitro maturation using standard procedures and then fixed and stained for assessment of nuclear maturation. Collectively, hormonal stimulation did not increase the number of ovarian antral follicles available compared to the control group (P > 0.05), but did result in higher output of medium (Group B: 9.8 +/- 7.1; Group C: 3.4 +/- 6.7; Group D: 15.6 +/- 4.9 versus Group A: 1.6 +/- 2.2) and large follicles (Group B: 44.8 +/- 22.9; Group C: 8.7 +/- 6.1; Group D: 70.2 +/- 10 versus Group A: 6.1 +/- 6.3). Administration of hCG 12 h before follicle aspiration proved to be the best strategy to obtain high numbers of immature and mature oocytes from antral follicles (P < 0.05; Group B: 70.8 +/- 12 and Group D: 82 +/- 12.6 versus Group A: 43.6 +/- 13.9 and Group C: 27.2 +/- 13.9). A significantly higher number of expanded COCs was obtained from hormonally stimulated groups compared to the control group (P < 0.05; Group B: 28.7 +/- 16.8, Group C: 16.3 +/- 5.9 and Group D: 27.1 +/- 16.9 versus Group A: 6.2 +/- 6). A higher oocyte maturational competence (P < 0.05) was found in Groups A, B and D (80.8 +/- 7.9, 87.5 +/- 8.2, and 86.5 +/- 4.3, respectively) compared to Group C (60 +/- 26.2). In conclusion, in prepuberal buffalo calves combined gonadotrophin stimulation protocols yielded higher numbers of medium to large size follicles compared to a control group. A high number of good quality oocytes were recovered by transvaginal ultrasound follicle aspiration, and a high rate of metaphase II progression was reached after in vivo and in vitro maturation.

Ovarian follicular dynamics and hormonal profiles in heifer and mixed-parity Mediterranean Italian buffaloes (Bubalus bubalis) following an estrus synchronization protocol.

The primary objective was to elucidate ovarian follicular dynamics and hormonal profiles in nulliparous heifer (HE; n = 11 ) and mixed-parity (MP; n=10 ) Mediterranean Italian water buffaloes (Bubalus bubalis) following an estrus synchronization protocol. Both groups received a progesterone releasing intravaginal device (PRID) implant for 10 days; a luteolytic dose of synthetic prostaglandin was given 7 days after PRID insertion. Daily ultrasound monitoring and collection of blood to determine plasma concentrations estradiol and progesterone started 1 day after PRID removal and lasted for 55 and 65 days in HE and MP buffaloes, respectively. Data analysis was restricted to the first 5 days after PRID removal and to one estrus cycle following induced ovulation. The HE buffaloes were not inseminated and only one ovulated within 5 days after PRID removal; the remainder ovulated between 8 and 48 days after PRID removal (except one in which ovulation was never detected). All HP buffaloes were inseminated 72, 96 and 120 h after PRID removal; seven buffaloes ovulated within 5 days after PRID removal and two were pregnant. Mean diameter of the largest follicle was significantly smaller in HE than MP buffaloes the first 4 days after PRID removal. There was a parity by time interaction ( P=0.0047 ) for plasma progesterone concentrations; progesterone was higher in HE than MP buffaloes 1 day after PRID removal, but the converse was true 2 days after PRID removal. After induced ovulation, HE buffaloes exhibited a one-wave ( n=5; length of cycle, 8-12 days), two-wave ( n=4; range: 20-26 days) or three-wave cycle ( n=1; 25 days). In contrast, all non-pregnant MP buffaloes ( n=8 ) had a two-wave cycle (range: 19-25 days). For buffaloes with two-wave cycles, the growth rate and diameter of the largest follicle was significantly smaller in HE than MP buffaloes for both the first follicular wave (1.3mm versus 1.7 mm per day and 10.5 mm versus 13.3 mm, respectively) and the second follicular wave (1.0 mm versus 1.3 mm per day and 11.0 mm versus 13.8 mm). In conclusion, there were many significant morphological and endocrine differences between HE and MP buffaloes.

Plasma estrone sulfate, clinical biochemistry, and milk yield of dairy cows carrying a fetus from a bull or its clone.

The aim of this article was to compare plasma estrone sulfate (E1SO4), clinical biochemistry, and milk yield of dairy cows carrying a female fetus from a bull (BULL) or from its clone (CLONE), evaluating also the relationship between the former variables and the birth weight of the newborn. Sixteen recipient dairy Friesian heifers (10 BULL and 7 CLONE) received a female embryo, obtained by in vitro embryo production and sexing by polymerase chain reaction with the semen of the BULL or the CLONE. Blood samples on all cows were obtained before feed distribution in the morning from jugular vein from 4 weeks before to 4 weeks after calving, to be analyzed for metabolic profile. The samples from late gestation were also analyzed for E1SO4 concentration. To separately assess the effect of calf birth weight (CBW), data were categorized as follows: low (<39 kg; BWT-A), mid (39-46 kg; BWT-B), and high (>46 kg; BWT-C). The plasma concentrations of ß-hydroxybutyric acid (BHB, P=0.019), Na (P=0.002), Cl (P=0.026), strong cation-anion balance (P=0.020), total bilirubin (P=0.054), and α1-globulin (P=0.044) were higher in prepartum BULL recipients than those in CLONE, whereas BHB (P=0.021) and Mg (P=0.090) were higher in postpartum BULL recipients, while no differences were recorded in the remaining postpartum parameters. The CBW class had significant interaction with week of gestation on antepartum plasma estrone sulfate (P=0.021), whereas CBW per se affected antepartum plasma BHB (P=0.021), and nonesterified fatty acids (NEFA; P=0.011) being higher in BWT-C which also had the lower NEFA concentration during postpartum. Milk yield was unaffected by the sire used, both for quantitative and qualitative aspects. Cows carrying heavier fetus (BWT-C) had a different lactation affected by month compared with the other 2 CBW groups. From these results, there were no differences between BULL and CLONE recipients. Estrone sulfate, BHB, and NEFA may be used to predict CBW and provide different nutritional management during gestation.

A novel route of transplantation of human cord blood stem cells in preimmune fetal sheep: the intracelomic cavity.

The intracelomic route for in utero hematopoietic stem cell transplantation was evaluated in preimmune fetal sheep and the engraftment characteristics were defined. Twelve twin ovine fetuses (gestational age: 40-45 days) received intracelomic transplants of human CD3-depleted (50 x 10(6) per lamb) or CD34-selected (1-2 x 10(5) per lamb) cord blood hematopoietic stem cells. Engraftment was evaluated from cell suspensions of the liver, spleen, bone marrow, and thymus by flow cytometry, cloning assays, and polymerase chain reaction (PCR) analyses of human beta2-microglobulin. Four fetuses (33%) aborted shortly after intracelomic transplantation and were not evaluable for engraftment. Engraftment was detected in four fetuses obtained from cesarean delivery on day 70 after transplantation of CD3-depleted cord blood cells. The degrees of engraftment in these four fetuses ranged from 6%-22% in the different organs (as revealed by antigenic analysis of human CD45 with flow cytometry). Three fetuses obtained after cesarean section at 102 (no. 435184) and 105 (no. 915293, no. 037568) days and one fetus delivered at term that received CD34-selected cord blood cells had human engraftment with 10%, 32%, 20%, and 10% CD45(+) cells in bone marrow, respectively. In six of eight fetuses evaluable for human engraftment, chimerism was confirmed by PCR analysis for human beta2-microglobulin, which also identified human cells in brain, spinal cord, heart, lung, and skeletal muscle. This preliminary study indicates that intracelomic transplantation of human hematopoietic stem cells in fetal lambs is feasible and effective in terms of hematopoietic engraftment.