Longitudinal ultrasound and endocrine evaluations were conducted in two adult female Sumatran rhinoceroses (Dicerorhinus sumatrensis) over a period of 12-22 months to learn more about their reproductive physiology. Rectal ultrasonography was conducted to monitor ovarian activity. Blood samples were collected and analysed for progesterone and LH, and faecal samples were analysed for progestin metabolites. One female showed cyclic ovarian activity during the study period, whereas the other female showed no evidence of ovarian activity. The cyclic Sumatran rhinoceros appeared to be an induced ovulator, the first of its kind reported within the Perrisodactyla. Ultrasound examinations of the ovaries revealed the formation of anovulatory haemorrhagic follicles when the animal was not mated. These follicles appeared to undergo varied degrees of luteinization that resulted in irregular faecal progestin profiles. When allowed to mate, the female showed a 21 day reproductive cycle that was reflected in both faecal progestin and serum progesterone profiles. The concentration of serum LH was baseline before mating, increased approximately 30-fold within 1-2 h of intromission and returned to baseline within 22 h. Ovulation occurred within 46 h of copulation. The female conceived three times during the study. Pregnancy was detected using ultrasonography 14-16 days after mating, and the concentration of both serum progesterone and faecal progestins remained high. Early embryogenesis appeared to be similar to that in horses. However, each pregnancy terminated unexpectedly within the first 3 months of gestation. This study demonstrates the important role that basic research and reproductive technology can play in developing a natural breeding programme for an endangered animal in captivity.

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The aim of this study was to quantify the content of lipid droplets in bovine oocytes and embryos from Bos indicus (Bi), Bos taurus (Bt) and Bos indicus × Bos taurus (Bi × Bt). Oocytes were aspirated post-mortem and subjected to in vitro maturation, in vitro fertilization and in vitro development; the medium employed at each stage (TCM-199, TALP, SOF) was supplemented with (i) serum replacement (SR), (ii) foetal calf serum (FCS) or (iii) oestrous cow serum (ECS). The structure and distribution of the lipid droplets were established using electron microscopy, but were quantified using an optical microscope on semi-fine toluidine blue-stained sections. The highest percentage of embryos corresponded to those produced with FCS and ECS, which differed from embryos generated with SR (p < 0.05). The highest percentage of morulae and the lowest percentage of blastocysts were obtained with the SR supplement (p < 0.05). The oocytes cultured in FCS demonstrated a higher number of lipid droplets compared to those cultured in SR and ECS (p < 0.05). Less accumulation of lipids was observed in embryos supplemented with SR. The lowest and highest numbers of lipid droplets in oocytes corresponded to the Bi and Bt strain, respectively. The lowest amount of lipid droplets in embryos was observed in Bi (p < 0.05). In conclusion, supplementation of the in vitro development culture medium (synthetic oviduct fluid) with a synthetic substitute serum produced similar results in terms of embryo development compared to those obtained with FCS, but a decreased degree of lipid droplet accumulation was observed in the in vitro-cultured embryos.

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A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

BackgroundIn order to improve ICSI, appropiate sperm selection and oocyte activation is necessary. The objective of the present study was to determine the efficiency of fertilization using ICSI with chemically activated ovine oocytes and sperm selected by swim up (SU) or swim up + zona pellucida (SU + ZP) binding.ResultsExperiment 1, 4–20 replicates with total 821 in vitro matured oocytes were chemically activated with ethanol, calcium ionophore or ionomycin, to determine oocyte activation (precense of one PN). Treatments showed similar results (54, 47, 42 %, respectively) but statistically differents (P < 0.05) than mechanical activated oocytes in sham, ICSI and sham injection (13, 25, 32 %, respectively) (10–17 replicates; n = 429).Experiment 2: Twelve ejaculates and 28 straws of semen were used (11–19 replicates). Sperm were selected by SU in BSA-TCM 199-H medium. A total of 2,294 fresh sperm and 2,760 from frozen-thawed semen were analyzed after SU or SU + ZP binding. Fresh sperm selected by SU showed acrosome reaction (AR) of 59 %, the sperm selected by SU + ZP binding increased AR to 91 %. In comparison, the AR of frozen-thawed sperm using SU or SU + ZP binding was 77 and 86 %, respectively (P < 0.05).Experiment 3: fertilization in 200 mechanical activativated oocytes (17 replicates) was 4 %, but fertilization increased in ethanol activated oocytes after ICSI (12-28 %) (5–6 replicates). When fresh sperm only selected by SU were injected to 123 oocytes, a fertilization rate (28 %) was achieved; in sperm selected by SU + ZP was 25 % (73 oocytes). In comparison, in frozen-thawed sperm selected by SU, fertilization was 13 % (70 oocytes), whereas sperm from SU + ZP binding displayed 12 % (51 oocytes) (P > 0.05).ConclusionsChemical activation induces higher ovine oocyte activation than mechanical activation. Ethanol slightly displays higher oocyte activation than calcium ionophore and ionomicine. Sperm selection with SU + ZP increased AR/A and AR/D rates in comparison with SU in fresh and frozen-thawed sperm. According to this, in terms of fertilization rates, chemical activation after ICSI increased oocyte PN formation compared to mechanical activation. Also, fresh sperm treated with SU and SU + ZP were significantly different than frozen-thawed sperm, but between sperm treatments no significant differences were obtained.

The use of reproductive technologies such as somatic cell nuclear transfer (SCNT) for avian species has been limited by the inability to visualise the pronucleus or pronuclei within the blastodisc or germinal disc region, respectively, primarily due to the opacity of the large, lipid filled yolk. The main objective in the present study was to assess a method for visualising and enucleating the avian ovum, a critical step in developing the capability for cloning birds. The method utilised in the present investigation was epi-fluorescence transmitted light (top-side UV) microscopy (EFTLM), also known as fluorescence/oblique or fluorescence/differential interference contrast (DIC) illumination, combined with vital staining and DNA visualisation techniques. The use of EFTLM combined with micromanipulation methods adapted from mammalian cloning procedures showed that the vitelline membrane of the avian ovum can be pierced and aspiration of the pronucleus, once visualised, can be performed without compromising the ovum's structure. Two approaches for domestic chicken ova collection, i.e., in vivo and in vitro, were utilised and ova recovery rates were compared. Based on a statistical analysis, i.e., Fisher's exact test, the results of the in vivo versus in vitro ovulation ova recovery methods were significantly different (P < 0.05), with the in vivo ovulation method yielding more viable intact ova. In conclusion, enucleating the avian ovum using EFTLM combined with vital staining, DNA visualisation, and micromanipulation techniques can be a feasible option for future avian cloning endeavours; although it will require further refinement to improve overall efficiency.