Chelation of trace elements in preservation medium influences the quality of boar spermatozoa during liquid preservation at 5°C for 4 weeks.

BACKGROUND: The addition of a metal chelator, ethylenediaminetetraacetic acid (EDTA), to semen extender has the purpose of capturing trace element ions. OBJECTIVE: This study was conducted to evaluate the effects of EDTA on the quality and in vitro fertilisability of liquid-preserved boar spermatozoa. METHODS: In Experiment 1, semen samples were preserved in the semen extender supplemented with 0, 3, 6, or 12 mM of Na-EDTA at 5 degree C for 4 weeks. In Experiment 2, semen samples were preserved in the extender supplemented with 3 mM of Na-EDTA, Ca-EDTA, or Zn-EDTA and without chelator EDTA. RESULTS: When Na-EDTA was used as a chelating substance in the extender, 3 mM was a most suitable concentration for sperm motility and viability after cold preservation. The supplementation of 3 mM Ca-EDTA had advantages regarding sperm motility, viability and plasma membrane integrity. CONCLUSION: Our findings indicate that 3 mM Ca-EDTA is the most suitable metal-chelating substance for the liquid preservation of boar semen.

Cell cycle synchronization of skin fibroblast cells in four species of family Felidae.

This study was examined whether the species of felid affects synchronization accuracy at the G0/G1 stage of the cell cycle and the occurrence of apoptosis by different protocols, such as serum starvation, confluent and roscovitine treatment. Skin fibroblast cells were obtained from the Asian golden cat, marbled cat, leopard and Siamese cat. The cells from each animal were treated with either serum starvation for 1-5 days, cell confluency-contact inhibition for 5 days or roscovitine at various concentrations (7.5-30 µm). Flow cytometric analysis revealed that serum starvation for 3 days provided the highest cell population arrested at the G0/G1 stage, irrespective of the felid species. In all species, 100% confluency gave a significantly higher percentage of cells arrested at the G0/G1 stage compared with the non-treated control cells. The effects of roscovitine treatment and the appropriate concentration on the rates of G0/G1 cells differed among the felid species. Serum starvation for more than 4 days in the marbled cat and Siamese cat and roscovitine treatment with 30 µm in the Asian golden cat and leopard increased the rates of apoptosis. In conclusion, different felid species responded to different methods of cell cycle synchronization. Asian golden cat and Siamese cat fibroblast cells were successfully synchronized to G0/G1 stage using the serum starvation and roscovitine treatment, whereas only confluency-contact inhibition treatment induced cell synchronization in the leopard. Moreover, these three methods did not successfully induce cell synchronization of the marbled cat. These findings may be valuable for preparing their donor cells for somatic cell nuclear transfer in the future.

Birth of kittens after the transfer of frozen-thawed embryos produced by intracytoplasmic sperm injection with spermatozoa collected from cryopreserved testicular tissue.

The aim of this study is to produce live kittens from oocytes fertilized by intracytoplasmic sperm injection (ICSI) with frozen/thawed testicular spermatozoa. Spermatozoa were collected from thawed testicular tissue and subsequently injected into in vitro matured cat oocytes. At 24 h post-ICSI, presumptive zygotes/cleaved embryos were treated with 10 µm forskolin for 24 h to reduce intracellular lipid content of embryos (delipidation). At 48 h after oocyte injection, cleaved embryos (2- to 8-cell stage) were frozen in 10% (v/v) ethylene glycol-based medium by a slow controlled rate method and stored in liquid nitrogen. To evaluate in vitro and in vivo developmental competence, frozen embryos were thawed and then cultured for 6 days (n = 155) or cultured for 2 h before transferred (n = 209) to hormonal (equine chorionic gonadotropin/hCG)-treated cat recipients. Cleavage frequency at day 2 after ICSI with frozen/thawed testicular spermatozoa was ~30%. The percentages of frozen/thawed embryos that developed to morula and blastocyst stage (on day 3 and day 6 of in vitro culture, respectively) were significantly lower than that of fresh ICSI embryos (22.6 vs 45.2% and 21.3 vs 38.7%, respectively; p < 0.05). However, no difference was found in the number of blastomeres between frozen/thawed (242.5 ± 43.1) and fresh (320.2 ± 28.1) blastocysts. Three of seven cat recipients were pregnant and one pregnant cat delivered two healthy kittens. This is the first report of the birth of kittens after the transfer of frozen-thawed embryos produced by ICSI with frozen/thawed testicular sperm.

Follicular dynamics and oestrous detection in Thai postpartum swamp buffaloes (Bubalus bubalis).

This study characterized follicular activity and oestrous behaviour from 5 to 9 days post-calving up to the 4th ovulation postpartum (pp) in 16 multiparous (range 2-7 parities) Thai swamp buffalo cows (Bubalus bubalis), aged 4-12 years and weighing from 432 to 676 kg. Ovarian follicular activity was examined by transrectal ultrasonography (TUS) every morning. Oestrous detection was performed twice daily by direct personal observation of behaviour and for presence of clear cervical mucus discharge and indirectly by video camera recording during 21 h/day. A follicular wave-like pattern was present before the 1st ovulation leading to short oestrous cycles. Growth rates and maximum diameters of the ovulatory follicles did not differ between the 1st and 4th ovulations. However, growth rate for non-ovulatory dominant follicles (DF) before the 1st ovulation was lower than for the ovulatory follicle (p<0.05). In addition, the diameter of all ovulatory follicles (14.3 ± 0.46 mm, n=39) was significantly larger (p < 0.01) than those of the preceding last but one non-ovulatory DF (10.8 ± 0.20 mm, n = 5), but similar to the last preceding non-ovulatory DF diameter (12.92 ± 0.96 mm, n = 14). Short oestrous cycles were most common between the 1st and 2nd ovulations (93.75%, 15/16 cows, 10.2 ± 0.38 days) decreasing in prevalence thereafter (50%, 3/6 buffaloes, 12.0 ± 1.53 days). Oestrous signs were relatively vague around the 1st ovulation pp to become more easily detectable thereafter. This study suggests that properly fed swamp buffaloes could be mated successfully within 2 months pp, at their 2nd spontaneous ovulation, provided oestrous detection is at least performed daily at 06:00-08:00 hour.

Maturation competence of swamp buffalo oocytes obtained by ovum pick-up and from slaughterhouse ovaries.

This study was designed with the final goal of improving in vitro embryo production in the Thai swamp buffalo (Bubalus bubalis carabensis). Oocytes were collected by ovum pick-up (OPU) from six non-lactating multiparous swamp buffalo twice per week for 10 consecutive sessions followed by once-weekly collection for 10 consecutive sessions without hormone stimulation. In addition, oocytes were collected from slaughterhouse ovaries that were classified as follows: ovaries from non-pregnant cows with a visible corpus luteum (NPCL); pregnant cows with a corpus luteum (P); and non-pregnant cows without a corpus luteum (NP). Follicles in each group of ovaries were categorized as small (2-4 mm), medium-sized (5-8 mm) or large follicles (≥ 9 mm). The quality of the oocytes was assessed by their capacity to undergo in vitro maturation. The total number of observed follicles per session (all sizes combined) was larger in the once-weekly OPU group compared with the twice-weekly OPU group. In particular, the numbers of small and large follicles were higher in the once-weekly OPU group (5.2 ± 0.7 and 0.9 ± 0.2, respectively) than in the twice-weekly OPU group (3.9 ± 0.5 and 0.5 ± 0.1). The number of medium-sized follicles did not differ between the groups. The percentages of oocytes with an abnormal spindle morphology were not different between oocytes from the twice-weekly (30.0%) and the once-weekly (28.6%) OPU groups. A higher percentage of oocytes obtained in vitro (49.5%) exhibited nuclear abnormalities compared with those obtained in vivo (≤34.8%) after in vitro maturation. In conclusion, oocytes can be successfully collected by OPU in the swamp buffalo, without hormonal pretreatment, and per week more good-quality oocytes can be collected by twice-weekly OPU. In addition, oocytes collected from slaughterhouse ovaries can be used with the reproductive status of the cow having no influence on the maturation competence of oocytes.

Y-chromosomal variation confirms independent domestications of swamp and river buffalo.

Y-chromosomal variation in the water buffalo was analysed by sequencing of DBY, ZFY and SRY gene segments. A clear separation of the paternal lineages of the river and swamp types parallels the differences between their maternal lineages and nuclear DNA. Sequence divergence was found to be comparable to the divergence of taurine cattle and zebu, and this divergence predated domestication, confirming that river and swamp buffalo originated from different wild populations. Within a sample of 23 Thai swamp buffaloes, we identified four haplotypes with different geographical distributions, two of which were shared by Thai wild buffaloes.

Effect of docosahexaenoic acid on quality of cryopreserved boar semen in different breeds.

During the cryopreservation process, the level of polyunsaturated fatty acids, especially docosahexaenoic acid (DHA), in the sperm plasma membrane decreases significantly because of lipid peroxidation, which may contribute to sperm loss quality (i.e. fertility) of frozen-thawed semen. The aim of this study was to investigate the effect of supplementation of DHA (fish oil) in freezing extender II on frozen-thawed semen quality. Semen from 20 boars of proven motility and morphology, were used in this study. Boar semen was split into four groups, in which the lactose-egg yolk (LEY) extender used to resuspend the centrifuged sperm pellet was supplemented with various levels of fish oil to reach DHA level of 1X (group I, control, no added fish oil), 6X (group II), 12X (group III) and 18X (group IV). Semen solutions were frozen by using a controlled rate freezer. After cryopreservation, frozen semen was thawed and evaluated for progressive motility, viability by using SYBR-14/Ethidiumhomodimer-1 (EthD-1) staining and acrosome integrity by using FITC-PNA/EthD-1 staining. There was a significantly higher (p < 0.001) percentage of progressive motility, viability and acrosome integrity in DHA (fish oil) supplemented groups than control group. Generally, there seemed to be a dose-dependent effect of DHA, with the highest percentage of progressive motility, viability and acrosome integrity in group-III. In conclusion, supplementation of the LEY extender with DHA by adding fish oil was effective for freezing boar semen as it resulted in higher post-thaw plasma membrane integrity and progressive motility.

On-farm lambing outcomes after transfer of vitrified and slow frozen embryos.

The aim of the present study was to determine the most suitable embryonic stage and embryo freezing technique for commercial implementation of frozen embryo trading by small-scale sheep producers. There was a 2 × 2 factorial design utilized for conducting the study consisting of two embryo stages (2-8 cells or morula/blastocyst) and two cryopreservation protocols (vitrification or slow-freezing). For the in vivo produced embryos, there were treatments of crossbred donor ewes to induce superovulation. Embryos were recovered surgically on either Day 2 or 5.5 after estrous onset. The embryos were cryopreserved using either a vitrification or slow-freezing method before there was transfer to recipients. Ovarian response, embryo survival and lambing outcomes were analyzed. There were no differences in number of recovered and fertilized embryos at the two embryonic developmental stages. There were no effects of embryonic stages and cryopreservation methods on pregnancy rate, twinning rate, fetal birth weights and lamb weight at 1 month of age. When there was use of vitrified embryos for transfers, there was a greater lamb weight at 2 months of age (8.38 ± 0.20 compared with 7.78 ± 0.21 kg; P = 0.044) than when there was transfer of embryos cryopreserved using slow freezing procedures. Considering economic and practical benefits to small-scale sheep farms, morula/blastocyst stage-embryo collection and transfer into the uterus is more efficacious than transferring 2-8 cells embryos into the oviduct. Results of this study may contribute to the genetic improvement in the flocks of small-scale sheep producers.

Light and scanning electron microscopic studies of oviductal epithelium in Thai swamp buffalo (Bubalus bubalis) at the follicular and luteal phases.

The purpose of this study was to investigate the morphological changes in the epithelium of Thai swamp buffalo oviducts at the follicular and luteal phases by histological technique and scanning electron microscopy. The samples from the infundibulum, ampulla, isthmus and uterotubal junction (UTJ) of the oviduct were taken immediately after slaughter at the local abattoir. Noticeable cyclic changes were observed on the epithelial surface of the infundibulum and ampulla, but few changes were present in the isthmus and UTJ. At the follicular phase, the epithelium of infundibulum and ampulla were densely covered with ciliated cells whose cilia concealed the apical processes of the secretory cells. In contrast, the secretory cells dominated in the epithelium at the luteal phase and most of the ciliated cells were hidden by the bulbous processes of these cells. In the isthmus and UTJ at the follicular and luteal phases, the secretory cells were almost flat or gently rounded and covered with numerous microvilli at their apical surface. In conclusion, the histological and ultrastructural observation of Thai swamp oviduct epithelium revealed marked cyclic changes in the cellular differences associated with the main functions of segmental variations.

Effects of straw volume and Equex-STM on boar sperm quality after cryopreservation.

The present experiments were designed to study the effect of adding the detergent Equex-STM to freezing extender, and of straw volume (0.25 ml vs 0.5 ml), on boar sperm quality after cryopreservation. Three ejaculates from each of four purebred boars (three Landrace and one Yorkshire) were collected and frozen with a lactose-egg yolk extender containing glycerol with or without 1.5% Equex-STM. The extended semen was loaded into either 0.25- or 0.5-ml straws. The straws were placed in liquid nitrogen (LN(2)) vapour approximately 3 cm above the level of LN(2) for 20 min and then were plunged into LN(2). Thawing was achieved in warm water at 50 degrees C for 12 s and then was incubated in a 38 degrees C water-bath for 30 min before evaluating sperm quality. Results showed that the individual motility, viability and acrosomal normal apical ridge (NAR) were improved (p < 0.001) when Equex-STM was added to the freezing extender. There was no difference (p = 0.48) in sperm motility between 0.25- and 0.5-ml straws when Equex-STM was added. The percentages of viable and of NAR sperm in 0.5-ml straws were higher than those in 0.25-ml straws (p = 0.02, p = 0.0003 respectively). The percentages of membrane intact sperm evaluated using the short hypo-osmotic swelling test were not affected by straw volume or the adding of Equex-STM (p > 0.05). The results of these investigations suggested that Equex-STM exerts a beneficial effect on the quality of cryopreserved boar semen and this cryopreservation protocol was favourable for a 0.5-ml straw.

The effect of antioxidants on motility, viability, acrosome integrity and DNA integrity of frozen-thawed epididymal cat spermatozoa.

Antioxidants partially ameliorated the negative effects of reactive oxygen species (ROS) produced during cryopreservation. The objective of the present study was to investigate the effect of cysteine and a water-soluble vitamin E analogue on the quality of frozen-thawed epididymal cat spermatozoa. Epididymal spermatozoa were collected from eight male cats and divided into three aliquots; these were resuspended with a tris egg yolk extender I (EE-I), or the same extender supplemented with 5mM dl-cysteine (EE-C) or with 5mM of a water-soluble vitamin E analogue (EE-Ve). Prior to the freezing step, sperm suspensions were added to the extender with Equex STM paste (EE-II). Sperm motility, progressive motility, membrane integrity, and acrosome status were evaluated at collection, after cooling, and at 0, 2, 4, and 6h post-thaw. Sperm DNA integrity was evaluated at 0 and 6h post-thaw. Relative to the control group, supplementation with vitamin E improved (P<0.05) post-thaw motility (69.4+/-5.6%), progressive motility (3.9+/-0.3), and membrane integrity (65.1+/-8.1%) immediately after thawing, whereas cysteine supplementation improved (P<0.05) post-thaw motility after 2h of incubation (53.8+/-12.2%) and DNA integrity after 6h (84.1+/-4.4%). However, neither antioxidant significantly increased the acrosome integrity of frozen-thawed spermatozoa. In conclusion, cysteine or vitamin E supplementation of tris egg yolk extender improved motility, progressive motility and integrity of the sperm membrane and DNA of frozen-thawed epididymal cat spermatozoa.

Reproductive performance of dairy cows with ovarian cysts after synchronizing ovulation using GnRH or hCG during the warm or cool period of the year.

This study was designed to compare the reproductive response to timed AI of lactating dairy cows with cystic ovarian follicles treated with GnRH or hCG to synchronize ovulation. The effectiveness of treatment during the warm or cool period of the year was also compared. Cows were given 12 microg GnRH-agonist i.m. on day 0 of the protocol, 15 mg PGF(2alpha) i.m. on day 7, and either GnRH-agonist (GPG treatment) or 3000 IU hCG i.m. (GPH treatment) on day 9, followed by timed AI. The cows were randomly chronologically assigned to GPG (n=130) or GPH (n=136) group. All cows were inseminated at fixed time 16-22 h after the end of treatment. During the warm period the pregnancy rate to first AI was 12% (7/60) and 21% (14/68) for the GPG and GPH groups, respectively, there being no significant differences between groups; the cumulative pregnancy rate was 22% (13/60) and 21% (14/68) for the GPG and GPH groups, respectively, again with no significant intergroup differences. During the cool period pregnancy rate to first AI was not different between groups: 29% (20/70) for GPG and 32% (22/68) for GPH, respectively; whereas the cumulative pregnancy rate was significantly higher (P<0.05) for the GPH groups than for the GPG group: 56% (39/70) and 78% (53/68), respectively. These findings indicate that during the warm period, the pregnancy rates of the cystic cows were similar whether they received GPG or GPH treatment, during the cool period, there is a beneficial effect to use hCG at day 9 of the ovsynch protocol compared GnRH on cumulative pregnancy rate.

Age, body weight and backfat thickness at first observed oestrus in crossbred Landrace x Yorkshire gilts, seasonal variations and their influence on subsequence reproductive performance.

The objective of the present study was to investigate puberty attainment in crossbred Landrace x Yorkshire (LY) gilts reared under tropical conditions and their subsequent reproductive performance. This study was carried out in a 2400-sow herd over a 1-year period. A total of 696 crossbred LY replacement gilts were included. Faecal samples from 214 gilts were collected to determine the faecal progesterone profiles around the time of first oestrus. Solid-phase 125I-radioimmunoassay was used to determine the progesterone concentrations in the faecal extract. The gilts entered the herd at an average age of 177.5 +/- 12.6 days, 95.7 +/- 10.2 kg body weight (BW) and a backfat thickness (BF) of 12.0 +/- 2.9 mm. On average, the gilts expressed first standing oestrus at 195 days of age, 106 kg of BW and a BF of 13.0 mm. The interval from entry to the gilt pool to the first observed oestrus (EOI) was 24.4 +/- 18.0 days (range 0-88 days). The hormonal profile indicated that the gilts that actually ovulated during the first observed oestrus was 34% (group A), the gilts that had ovulated before the first observed oestrus was 21% (group B) and the gilts that did not ovulate during the first observed oestrus was 45% (group C). During summer the proportion of group A gilts was significantly lower than during the winter and the rainy seasons (P < 0.05). The BW of gilts at entry significantly correlated with the BF at entry (r = 0.31, P < 0.001), the age at entry (r = 0.47, P < 0.001), the BW at first oestrus (r = 0.65, P < 0.001) and the BF at first oestrus (r = 0.33, P < 0.001). An increase of BW at entry of 1 kg resulted in a decrease of EOI of 0.28 days. The age, BW and BF of gilts at the first observed oestrus significantly influenced the total number of piglets born per litter (TB) and the number of piglets born alive per litter (BA) in the first three parities. Gilts expressing their first oestrus between 181 and 200 days had a significantly larger TB than gilts that expressed first oestrus between 150 and 180 days (P = 0.03) and between 201 and 220 days (P = 0.003). Gilts that showed first oestrus between 110.1 and 120.0 kg had a larger TB and BA than gilts that showed first oestrus between 80.0 and 100.0 kg (P < 0.05). Gilts that showed first oestrus with a BF between 13.1 and 15.0 mm had a larger TB and BA than gilts that showed first oestrus with a BF between 11.1 and 13.0 mm (P < 0.05). Group A gilts had a significantly larger TB than group B (10.5 piglets/L versus 9.4 piglets/L, P = 0.02), while farrowing rate (FR) did not differ significantly among groups A, B and C (78.1, 76.9 and 77.6%, respectively). Gilts that farrowed in the summer had a larger TB and BA than gilts that farrowed in the winter (TB, P = 0.03; BA, P = 0.09) and the rainy season (TB, P = 0.006; BA, P = 0.003). In conclusion, LY gilts reared under tropical conditions expressed first standing oestrus at 195 days of age, 106 kg BW and a BF of 13.0 mm. Under field conditions, 21% of the gilts with an observed oestrus had ovulated. The proportion of gilts that showed first oestrus and ovulated normally was lowest during the summer. The age, BW and BF at first observed oestrus influenced subsequent reproductive performance over the first three parities. The mean litter size (TB and BA) in the first three parities were highest in gilts that had a first observed oestrus between 181 and 200 days with 110.1-120.0 kg BW and 13.1-15.0 mm BF.

Population genetic analysis and genome-wide association study of patellar luxation in a Thai population of Pomeranian dogs.

The genetics of patellar luxation (PL) were investigated in Pomeranian dogs presented at the Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University. A cohort of 339 Pomeranian dogs, part of a four-generation pedigree of 842 Pomeranians, was screened for PL from 2006 to 2013. PL was present in 77% of the screened dogs, with 84% having bilateral and 16% unilateral luxation. Medial PL was more common (95%) than lateral PL (2%) or bidirectional PL (3%). The risk of PL was similar in male and female dogs (female:male relative risk 1.11, 95% CI 0.98-1.25). The heritability of PL in the screened population was 0.44±0.04 using a threshold model. A genome-wide association study of PL (48 cases and 48 controls) using a high-density SNP array indicated the possible involvement of 15 chromosomal regions, of which CFA05 and CFA32 remained associated in a larger study involving an additional 128 cases and 7 controls. Candidate genes in these regions may be involved in the pathogenesis of PL in Pomeranian dogs.

Relationships between ovulation rate and litter size in purebred Landrace and Yorkshire gilts.

The aim of the present study was to investigate the ovulation rate and its relationship to number of total piglets born in purebred gilts under tropical climatic conditions. This study was conducted in two swine breeding herds (A and B) in the northeastern part of Thailand. The sources of swine genetic material originate from West Europe. Gilts were mated (AI) on the second or later observed estrus at a body weight of at least 130 kg. In most cases, they were mated at third estrus. One hundred and twenty-seven gilts, 24 Landrace and 24 Yorkshire from herd A, and 42 Landrace and 37 Yorkshire from herd B were used. Gilts were examined once by laparoscopy under general anesthesia between days 8 and 15 after mating. The ovaries were examined and the pathological findings were recorded. The number of corpora lutea was counted, and was assumed to equal the ovulation rate. Subsequent mating results and farrowing data were recorded. The data were analyzed with analysis of variance. Single or double unilateral cysts and par-ovarian cysts did not affect mating results. Landrace gilts were significantly younger at first mating than Yorkshire gilts (244 versus 249 days, P < 0.05). At first mating, Yorkshire gilts had a significantly higher ovulation rate compared to Landrace gilts (15.3 versus 13.8, P < 0.001). There was no difference in the number of total piglets born per litter between the two breeds, but the total prenatal loss from ovulation to farrowing was significantly higher in Yorkshire than in Landrace gilts. Both the low ovulation rate and the high prenatal loss contribute to the low litter size in gilts raised under tropical climatic conditions.

Ovarian responses and oocyte recovery in prepubertal swamp buffalo (Bubalus bubalis) calves after FSH or PMSG treatment.

Stimulation of follicular growth was examined using two different gonadotropin treatments in 10 prepubertal swamp buffalo calves (8 to 12 mo old). Each calf received an ear implant consisting of 3 mg norgestromet and 5 mg estradiol valerate during hormonal treatment. Five calves were additionally administered FSH (24 mg, im) and, 2 mo later, PMSG (3,000 IU). The remaining 5 calves were first treated with PMSG followed by FSH. Ovarian responses to treatments were examined by laparotomy, 72 h after ear implant removal, and by the number of follicles (diameter > or = 0.8 cm) and corpora hemorrhagica present. Ovaries had more significant response to FSH than PMSG treatment (13.9+/-8.6 vs 5.9+/-3.3 follicles; P<0.01). Although the recovery rate tended to be lower for FSH treated (64%) than PMSG-treated (82%) animals, more oocytes/animal were harvested in the PMSG treatment (8.3+/-5.0 vs 4.6+/-3.2, respectively). The immature oocytes (n = 38) were cultured for 24 to 25 h in maturation medium (TCM-199 NaHCO3+10% fetal calf serum [FCS] in 5%CO2 in air at 39 degrees C). Oocyte maturation was assessed after fixation and staining with aceto orcein. The in vitro maturation rate was 52.6% (20/38). This study shows the possibility of harvesting oocytes from prepubertal swamp buffalo calves and maturing the oocyte in vitro.

Early embryonic development in Thai swamp buffalo (Bubalus bubalis ).

A total of 33 nonsurgical embryo collections was carried out to investigate early embryo development in Thai swamp buffalo. Collections were performed on Days 5.5, 6.0, 6.5, 7.0 and 7.5. The different stages of embryo development on these days were the 16-cell stage, compact morula, blastocyst, hatched blastocyst and hatched expanding blastocyst, respectively. In addition, some degenerating embryos and unfertilized ova were also recovered. A higher recovery rate was obtained with single embryo collection after natural estrus than after induced estrus or superovulation, 78% (7 9 ) vs 46% (6 13 ) vs 54.5% (6 11 ), respectively. A higher percentage of normal embryos was also obtained with single embryo collection after either natural or induced estrus than after superovulation, 71% (5 7 ), 83% (5 6 ) and 38% (6 16 ), respectively.

The effect of gonadotropin releasing hormone on superovulation in elite swamp buffalo cows (Bubalus bubalis).

The effect of gonadotropin releasing hormone (GnRH) supplement was investigated in twenty eight FSH-treated buffalo cows. Animals were assigned to three groups; Group I: GnRH was given at standing heat (n=9), Group II: GnRH was given 8-12 hr after standing heat (n=8) and Group III: Control group with FSH alone (n=11). The responses (no. of corpora lutea and no. of anovulatory follicles), the number of recovered embryos and transferable embryos among the three groups were compared following slaughter of the animals on days 6 to 7 after first mating. The results indicated that the application of GnRH in FSH-treated animals gave no advantage by increasing in the number of ovulations or recovered embryos in all the treatment groups (P>0.05): 4.33 +/- 3.35 vs 3.88 +/- 4.09 vs 4.5 +/- 2.68 for corpora lutea, and 2.33 +/- 2.24 vs 2.0 +/- 3.20 vs 1.91 +/- 2.74 for recovered embryos respectively. GnRH treatment tended to reduce the number of anovulatory follicles but the finding was not significant; 6.11 +/- 3.3 vs 7.38 +/- 4.84 vs 10.18 +/- 2.74 follilcles (P>0.05). The supplementation of GnRH at 8-12 hr after standing heat seemed to produce more transferable embryos than those of treated at standing heat or the controls 1.63 +/- 2.77 vs 1.25 +/- 1.67 vs 1.36 +/- 1.69 embryos respectively.

The effect of GnRH supplement of FSH and PMSG treatments for prepubertal swamp buffalo calves (Bubalus bubalis).

The objective of the experiment was to improve the multifollicle stimulation technique and the ovarian response examination in prepubertal swamp buffalo calves. Six animals were stimulated by gonadotropin hormone 7 days after a progesterone ear-implant. The first stimulation was done by giving 24 mg FSH + 100 microg GnRH (FSH+GnRH) and the second, one month after by giving 2,000 IU PMSG + 100 microg GnRH (PMSG+GnRH). Twenty-four hours after GnRH, the ovarian responses were checked using rectal palpation and real-time B mode ultrasonography. Five out of six animals (83.3%) responded to both treatments and were selected for oocyte collection. The oocytes were aspirated directly following a caudal midline laparotomy. The results of ovarian responses to FSH+GnRH and PMSG+GnRH averaged 17.6+/-12.1 (L-9.8+/-8.7, R=7.8+/-6.2) and 17.4+/-5.6 (L-9.4+/-2.9, R=8.0+/-3.7), respectively. The average number of recovered oocytes per animal was 9.0+/-6.4 and 8.4+/-1.1, respectively which represented a recovery rate of 56.3 (+/- 9.2)% and 51.9 (+/- 10.3)%. More than eighty percent of the recovered oocytes were in an immature stage with more than 2-3 layers of compact cumulus mass. The present study showed that the oocytes were collected successfully in prepubertal buffalo calves after the FSH+GnRH or PMSG+GnRH stimulation and most of the recovered oocytes were immature, which made them suitable for in vitro maturation and fertilization.

First cloned swamp buffalo produced from adult ear fibroblast cell.

The world's first cloned swamp buffalo (Bubalus bubalis) derived from adult ear skin fibroblast has been reported. Donor fibroblast cells were produced from biopsies taken from adult male ear skin and in vitro matured oocytes obtained from a slaughterhouse were used as cytoplasts. A total of 39 blastocysts and 19 morulae fresh embryos were transferred into 12 recipient buffaloes. Progesterone assays indicated establishment of pregnancy in 10 of the 12 buffaloes (83.3%) after 45 days, with six animals still pregnant at 3 months. One recipient maintained pregnancy to term and naturally delivered a 40 kg male calf after 326 days of gestation. DNA analysis showed that the cloned calf was genetically identical to the donor cells. Genotype analyses, using 12 buffalo microsatellite markers, confirmed that the cloned calf was derived from the donor cell lines. In conclusion, the present study reports, for the first time, the establishment of pregnancy and birth of the first cloned Thai swamp buffalo derived from adult ear skin fibroblast cells.