Banteng and Bali cattle in Indonesia: status and forecasts.

Bali cattle still represents 27% of the total cattle population in Indonesia, and it is considered the pillar breed for small farmers. Moreover, it is a breed of evolutionary importance regarding its direct ancestry from Banteng. However, there is a need for the establishment of a rational system for the evaluation of breeding soundness for indigenous Bali bulls to be used as sires for artificial insemination breeding programmes. Moreover, there is a need for cryobanking of well-identified genetic resources pertaining their use in evolutionary research and application as essential germplasm in breeding programmes.

The origin of Indonesian cattle and conservation genetics of the Bali cattle breed.

Both Bos indicus (zebu) and Bos javanicus (banteng) contribute to the Indonesian indigenous livestock, which is supposedly of a mixed species origin, not by direct breeding but by secondary cross-breeding. Here, the analysis of mitochondrial, Y-chromosomal and microsatellite DNA showed banteng introgression of 10-16% in Indonesian zebu breeds with East-Javanese Madura and Galekan cattle having higher levels of autosomal banteng introgression (20-30%) and combine a zebu paternal lineage with a predominant (Madura) or even complete (Galekan) maternal banteng origin. Two Madura bulls carried taurine Y-chromosomal haplotypes, presumably of French Limousin origin. There was no evidence for zebu introgression in five populations of the Bali cattle, a domestic form of the banteng.

Individual variation in fresh and frozen semen of Bali bulls (Bos sondaicus).

AIM: This study aimed to analyze the individual factors influencing the sperm quality of Bali bulls at Baturiti Artificial Insemination (AI) center. MATERIALS AND METHODS: Semen that was ejaculated from nine Bali bulls was collected using artificial vaginas (n=5/bull). Semen ejaculates were evaluated immediately after collection to measure the quality of the fresh semen, including semen volume, sperm concentration, progressive motility, membrane integrity (MI), and abnormal morphology. Frozen semen was evaluated for progressive sperm motility, concentration, viability, MI, abnormal morphology, and deoxyribonucleic acid (DNA) fragmentation. Other secondary data, focusing on semen quantity (semen volume and sperm concentration), were also collected from frozen the semen production data of the Baturiti AI center from 2017 to 2019. Data were analyzed statistically using a completely randomized design, and one-way analysis of variance was applied to find differences among individual bulls. RESULTS: Significant differences (p<0.05) were found among the bulls in semen volume, sperm motility, concentration, and MI of the fresh semen. Significant differences (p<0.05) were also found among the bulls in sperm motility, viability, MI, abnormal morphology, and DNA fragmentation of the frozen semen. CONCLUSION: Individual variation in all the tested sperm parameters of the fresh semen of Bali bulls, except sperm viability and abnormalities, was noted. Similarly, individual variation in all the tested sperm parameters in frozen semen, except sperm concentration, was noted. Therefore, individual factors can be used for selecting a superior bull in Bali cattle.

Follicular dynamics prior to and during superovulation in heifers.

The present ultrasonographic study examined the relationship between certain follicular parameters and the superovulatory response in gonadotropin-stimulated heifers. Thirty heifers received a total of 35 mg FSH twice daily for 4 d and 0.75 mg cloprostenol were given to induce luteolysis and estrus at 72 h after the initial FSH injection. Transrectal ultrasonography was performed once daily from 1 or 2 d before the initial FSH injection and until the day of estrus. The number of small (2 to 4 mm), medium (5 to 9 mm), and large (>/=10 mm) size follicles as well as the diameter of the large follicles were recorded. Embryos were recovered non-surgically 6 or 7 d after estrus, and the number of corpora lutea was determined by palpation per rectum. Heifers with >2 or 0.05). The number of large follicles and the sum of medium and large follicles were positively correlated (r=0.43 and r=0.54, respectively; P<0.05) with the number of corpora lutea palpated on the day of embryo recovery (6 to 7 d after estrus). In conclusion, there was an effect of the day relative to initiation of FSH treatment on all follicular categories in heifers responding positively to superovulation, and there was no effect of side (left or right ovary) or of corpus luteum diameter (ipsilateral or contralateral).

The freezability of biopsied bovine embryos.

A biopsy of 1-5 blastomeres was taken from 86 bovine compacted morulae, using a technique, that did minimal damage to the zona pellucida. As soon as possible after the micromanipulation the embryos were frozen, without sealing the penetrated zona pellucida. In order to evaluate the viability of the biopsied frozen-thawed embryos, half of them were transferred to synchronized recipients while the remaining half were co-cultured to evaluate the developmental capacity. A control group of 43 intact embryos was subjected to the same procedures. This study revealed a slight, but not significant, decrease in the pregnancy rate of the biopsied embryos after freezing and thawing, although the embryos by co-culture with bovine oviduct epithelial cells revealed normal morphology and developmental rate. It is concluded that the described biopsy technique did not compromise the freezability of 7-day-old bovine embryos.

Follicular development and embryo recovery following 3 versus 8 FSH injections in heifers.

Ovarian follicular dynamics and embryo yield were studied during 2 different FSH regimens for superovulation of cattle. Twenty heifers were given intramuscular injections of FSH (total of 35 mg NIH) either once daily for 3 days (Group 3x1) or twice daily for 4 days (Group 4x2). At 72 h after the first FSH injection, each animal was injected with 0.75 mg cloprostenol. Inseminations were performed at 12 h and 24 h after the onset of heat. Transrectal ultrasonography was performed on the day of the first FSH injection, the day of cloprostenol injection, the day of insemination and finally on the day of embryo recovery (day 6 or 7 after heat). The numbers of small (2-4 mm), medium (5-9 mm) and large (> 10 mm) size follicles were recorded. The total number of corpora lutea, eggs and transferable embryos were recorded on the day of embryo recovery. No differences were found between the 2 groups in either of the parameters studied (p > 0.05). It can be concluded that treatment with this FSH preparation once daily for 3 days gives a folliculogenic and superovulatory response similar to a treatment regimen where it is given twice daily for 4 days.

Oviduct epithelial cell co-culture of early porcine embryos.

One- to 16-cell porcine embryos were cultured in either Whittens medium supplemented with bovine serum albumin and fetal calf serum (WM) or in the same medium with porcine oviduct epithelial cell co-culture (WM-Poec). All stages of embryos cultured in WM-POEC had higher cell counts after 144-168 h of development than did embryos in WM. There was however, no significant difference in blastocyst formation rate of embryos cultured in WM-POEC over those cultured in WM. A high proportion of the embryos entering culture at the 1-2-cell were able to pass the 4-cell block stage in both WM and WM-POEC, 81% and 77%, respectively. In both media, most of the 1-2-cell embryos arrested their development at the compacted morula stage and failed to blastulate while embryos initiating culture at the 4- and 8-16-cell embryos formed blastocysts in culture at a rate of 80-90%.

Coculture of bovine demiembryos prior to freezing.

Pregnancy rates after transfer of frozen/thawed bisected embryos, demiembryos, have until now been very low. In the present study it was attempted to improve the freezability rate of demiembryos by culturing them on a monolayer of bovine oviduct epithelial cells (BOEC) prior to freezing. The cultured frozen/thawed demiembryos showed a lower developmental rate than intact embryos. The pregnancy rate (23%) following transfer was not different from the pregnancy rate after transfer of unfrozen demiembryos (26%). The calving rate (4%), however, was significantly lower than the calving rate after transfer of fresh demiembryos (23%). Although the overall pregnancy rate achieved in this study was low, it can be concluded that a co-culture period upon BOEC prior to freezing does not improve the viability of frozen demiembryos.

Blastomere content of cultured/frozen bovine demiembryos.

The purpose of the study was to evaluate whether a period of co-culture with bovine oviduct epithelial cells (BOEC) could improve the tolerance of bisected bovine embryos to freezing and thawing. Day 6 embryos were bisected and the resulting demiembryos were stained with Hoechst 33342 and cell counts were made by counting intact blastomere nuclei. Of these, 11 were stained as freshly manufactured demiembryos, 25 after co-culture for 24 h with BOEC and 37 stained after 24 h co-culture and freezing and thawing. The staining revealed, that there was no significant difference in cell count of demiembryos that were stained immediately after bisection, compared to those, that were co-cultured for a 24 h period. Also, the co-cultured/frozen/thawed demiembryos had a significant decrease in cell numbers compared to the non-frozen demiembryos. We conclude, that a 24 h period of co-culture with BOEC does not result in appreciable cellular proliferation in demiembryos and therefore instead of improving the survival of frozen/thawed demiembryos by giving them opportunity to multiply their cell number and thus make them more resistant to cell damage, rather compromised the viability of cryopreserved demiembryos.

Oocyte morphology in dominant and subordinate follicles.

The structure of oocytes aspirated from the dominant and its subordinate follicles was investigated from the achievement of follicular dominance to ovulation. Ovulation was induced in 18 heifers and 5 cows by injection of cloprostenol at days 8-14 (day 0 = day of ovulation), and follicular development was monitored by ultrasonography. The animals were slaughtered at days 3-11, but animals slaughtered on days 8-11 were given a second injection of cloprostenol at day 7 to allow ovulation of the dominant follicle of the first follicular wave. Oocytes were aspirated from the dominant (largest) and two largest subordinate follicles and processed for transmission electron microscopy, whereas the follicular fluids were analyzed for concentrations of estradiol-17 beta (E2) and progesterone (P4). Dominant follicular growth was associated with increase in the concentration of E2 and P4 in the follicular fluid, which was E2-dominated. From days 3-7, the dominant oocytes had pronounced junctional contacts with the cumulus cells and a nonundulating nuclear envelope but showed an increase in the number of lipid droplets and a decrease in the size of Golgi complexes, the size of cortical granule clusters, and the number of microvilli stacks. After cloprostenol injection on day 7, but before the anticipated LH surge, the dominant oocytes showed a reduced oocyte-cumulus contact, vacuolization of the nucleolus, undulation of the nuclear envelope, and dispersal of the mitochondrial clusters. The morphological alterations occurring in the dominant oocytes before the anticipated LH surge are suggested to be a prerequisite for the oocyte to achieve the competence to undergo final maturation. Subordinate follicles ceased growing at about days 3-4 and their follicular fluid had low E2:P4 ratio or was P4-dominated. Subordinate oocytes displayed degenerative features in their cumulus investment and nuclear activation and maturation especially after day 5. The structural changes associated with oocyte degeneration showed similarities with the processes seen before and during final maturation of the dominant oocytes.