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.

Improved in vitro development of OPU-derived bovine (Bos taurus) embryos by group culture with agarose-embedded helper embryos.

The average number of oocytes collected by ovum pick up (OPU) from Bos taurus cattle is <8 per live donor. The objective was to determine whether development of small numbers of cattle embryos (produced by OPU and IVF), was enhanced by including "helper" embryos, produced from abbatoir-derived oocytes and embedded in agarose. Oocytes were from abbatoir-derived ovaries (Experiments 1 and 2) or OPU of elite donors (Experiment 3). In Experiment 1, cleaved embryos (2-8 cells), were randomly allocated. Controls were groups of 1, 3, 5, 10, and 20 cleaved embryos cultured in 50 µL serum-free SOF, whereas treatments were groups of 1, 3, and 5 embryos freely cultured along with helpers in groups of either 9, 7 or 5 embedded in agarose per droplet. Therefore, there were 10 cleaved embryos per droplet in combinations of 1 + 9, 3 + 7 or 5 + 5 (free + helper), respectively. There was an increase in the progression to blastocyst for 1-5 embryos per droplet, compared to 10 and 20 (6.6-24.2% vs. 39.2-43.3%, P < 0.05). For the tested free embryos, those cultured with helpers had increased blastocyst development over their control counterparts (39.3-49.5% vs. 6.6-24.2%, P < 0.05). When the number of embryos per droplet was 10 or 20, blastocyst percentage was similar (39.2-49.5%, P > 0.05). In Experiment 2, addition of an agarose chip to the culture medium did not significantly affect development to the blastocyst stage. In Experiment 3, after fertilizing OPU oocytes with sorted X-sperm, a group of three cleaved embryos were cultured in a droplet with either seven helpers (3 + 7) or alone (3 + 0). Blastocyst development of OPU oocytes in the 3 + 7 group was 37.1%, higher than that in the 3 + 0 group (11.8%, P < 0.05). In conclusion, limited numbers of OPU/IVF oocytes had competent development when cultured with helpers (embedded in agarose to provide physical separation).

Follicle turnover and pregnancy rates following oestrus synchronization protocols in Mediterranean Italian buffaloes (Bubalus bubalis).

An ultrasound assessment of follicle turnover following two different protocols for synchronization of oestrus and ovulation, as well as an assessment of achieved synchronization between ovulation and AI and conception rates in nulliparous and pluriparous buffaloes were carried out during months of increasing day length. Nulliparous buffaloes (n = 30) were subjected only to Ovsynch protocol whereas pluriparous buffaloes (n = 31) were assigned to Ovsynch (n = 14) or to PRID-pregnant mare serum gonadotrophin (PMSG) (n = 17) protocol according to the presence of functional CL confirming cyclic and acyclic conditions. Ultrasound examination of ovarian follicular dynamics at critical days in the course of synchronization treatments was employed to monitor the fate of the largest available follicles at the beginning of treatments. Such available dominant follicle would persist throughout the protocol as ovulating follicle (no-follicle shift) or would regress giving way to a new follicle to become dominant and ovulate (follicle shift). Furthermore, ultrasound monitoring would determine the degree of synchronization of ovulation and final outcome represented by pregnancy rates. Pregnancy rate following Ovsynch protocol was 40% (12/30) and 42.8% (6/14) in nulliparous and pluriparous buffaloes respectively (p = 0.8575). Most ovulations were synchronized and recorded at AI and the following day in nulliparous (24/30; 80%) and pluriparous (12/14; 85.7%) buffaloes respectively (p = 1.000). A follicle shift was recorded in 14 of 30 (46.6%) and 11 of 14 (78.5%) in nulliparous and pluriparous buffaloes respectively (p = 0.0466). Among established pregnancies: eight derived from follicle shift (66.6%) and four from no-follicle shift (33.3%) in nulliparous buffaloes, p = 0.0729 whereas in pluriparous buffaloes five (83.3%) derived from follicle shift and one from no-follicle shift (16.6%), p = 0.6154. Collectively, from 18 pregnancies in nulliparous and pluriparous buffaloes following Ovsynch protocol, 13 derived from follicle shift (72.2%) and five from no-follicle shift (27.7%), p = 0.0860. Pregnancy rate in pluriparous buffaloes following PRID-PMSG protocol was 70.5% (12/17). The majority of ovulations were synchronized and recorded at first, second AI and following day (13/17; 76.4%). A follicle shift occurred in 15/17 buffaloes (88.2%) and among the 12 recorded pregnancies, 11 derived from follicle shift (91.6%), p = 0.5147. In conclusion, pregnancy rates following Ovsynch protocol were similar in nulliparous and pluriparous cyclic buffaloes. A progestagen treatment on acyclic buffaloes but still displaying some ovarian follicular dynamics, resulted in significantly higher pregnancy rate compared with Ovsynch (p = 0.0376). According to the time of scheduled AI, a high degree of synchronized ovulations were recorded following the implementation of both protocols.

First established pregnancies in Mediterranean Italian buffaloes (Bubalus bubalis) following deposition of sexed spermatozoa near the utero tubal junction.

At the time of AI following Ovsynch protocol, a total of 51 buffaloes were randomly divided in a first group (n = 30) subjected to conventional AI into the uterine body with 20 million non-sex sorted frozen-thawed spermatozoa, while a second group (n = 21) was inseminated near the utero-tubal junction (UTJ) ipsilateral to the ovary carrying the preovulatory follicle with 2.5 million live (4 million total) sex-sorted frozen-thawed spermatozoa. The semen used for flowcytometric sorting was collected and processed on a farm in Italy, and then shipped to a laboratory in Germany. Eleven buffaloes were inseminated with X-chromosome bearing spermatozoa and 10 with Y-chromosome bearing spermatozoa. Conception rates after conventional and UTJ inseminations were 43.3% (n = 13) and 42.8% (n = 9) respectively (p = 0.97). Eight of the nine foetuses obtained after insemination with sexed spermatozoa corresponded to the sex as predicted by the cell sorting procedure (five male and four female foetuses by ultrasound vs six male and three female foetuses by cell sorting). In conclusion, for the first time buffalo semen has been successfully subjected to procedures for flowcytometric sperm sorting and freezing. Low doses of sexed spermatozoa have been deposited near the UTJ giving conception rates similar to those of conventional AI with full dose.

Effect of recombinant bovine somatotropin and calcium salts of long-chain fatty acids on milk from Italian buffalo.

Fifty-one lactating Italian river buffalo were used in an 84-d study to evaluate the effects of recombinant bovine somatotropin (bST) and Ca salts of long-chain fatty acids on productive performance. Treatments were 1) control diet, 2) the control diet plus 0.3 kg/d of added Ca salts of long-chain fatty acids, 3) the control diet plus 320 mg of recombinant bST injected every 21 d for four cycles, and 4) the control diet plus 0.3 kg/d of added Ca salts of long-chain fatty acids and 320 mg of recombinant bST administered as previously described. Administration of bST and Ca salts of long-chain fatty acids increased milk production. Milk fat percentage was not affected by treatments. The percentage of short-chain fatty acids in milk fat was reduced by the addition of Ca salts. Medium-chain, long-chain, and unsaturated fatty acids in milk fat were increased by bST treatment. Milk protein percentage was decreased by the addition of Ca salts of long-chain fatty acids. Milk casein content, as a percentage of total protein or as a percentage of true protein, was unaffected by bST. Body condition score was lowered by bST administration, but the addition of Ca salts of long-chain fatty acids reduced body condition loss in buffalo that were treated with somatotropin.