Buserelin Research Articles

Progesterone-based timed AI protocols for Bos indicus cattle III: Comparison of protocol lengths

This study aimed to validate a 7 d progesterone (P4)-based fixed-time AI (FTAI) protocol for Bos indicus cattle by comparing to 8 and 9 d-type protocols. The first study compared 7 vs. 8 d protocols in Nelore heifers (Exp. 1.1; n = 742) and cows (Exp. 1.2; n = 2488), and the second study compared 7 vs. 9 d protocols in cows (Exp. 2; n = 1343). On experimental Day −10 and Day −11 the 8 and 9 d groups received an intravaginal P4 implant, 2.0 mg estradiol benzoate (EB) and 0.5 mg cloprostenol sodium (PGF). On Day −9 the 7 d group received the same treatments (P4, EB, and PGF). Then, on Day −2 all groups had the P4 implants removed, and PGF, 0.6 mg estradiol cypionate, and 300 IU equine chorionic gonadotropin (eCG) was administered. Fixed-time AI was performed 48 h later (Day 0) and 8.4 mg buserelin acetate (GnRH) was administered to 7d-G, 8d-G and 9d-G groups, whereas 7d-0, 8d-0 and 9d-0 groups did not receive GnRH at AI. Estrus was detected using tail-chalk between Day −2 and Day 0. Pregnancy per AI (P/AI) was evaluated by ultrasound 30 d after AI. Effects were considered significant when P ≤ 0.05, whereas a tendency was designated when P ≤ 0.10 and P > 0.05. In heifers (Exp. 1.1), incidence of estrus was similar regardless of protocol length (7 or 8 d). There was no independent treatment effect on P/AI or interaction between protocol length and GnRH at AI for P/AI (7d-0: 46.9, 7d-G: 51.4, 8d-0: 47.7, and 8d-G: 43.6%). Heifers in estrus had greater P/AI, and GnRH had no additional effect. More cows (Exp. 1.2) from the 8 d protocol were in estrus than cows submitted to the 7 d protocol. Additionally, despite no interaction between protocol length and GnRH on P/AI (7d-0: 55.9, 7d-G: 60.9, 8d-0: 56.2, and 8d-G: 60.8%), GnRH at AI increased P/AI. There was no interaction between estrus and GnRH, but cows displaying estrus had greater P/AI. Cows not expressing estrus tended (P = 0.06) to have greater P/AI when receiving GnRH. In Exp. 2, more 9 d cows were in estrus than 7 d cows. Protocol length did not affect P/AI but tended (P = 0.08) to interact with GnRH (7d-G had greater P/AI [57.9%] than 7d-0 [47.6%], but 9d-0 [54.6%] and 9d-G [55.4%] were not different from other groups). Moreover, GnRH increased P/AI only for the 7 d protocol. No interaction between estrus and GnRH was detected but estrus improved P/AI, and GnRH tended (P = 0.09) to improve P/AI of cows in estrus. In conclusion, despite longer protocols being more conducive to expression of estrus, there were no detectable effects of protocol length on P/AI. In addition, GnRH at FTAI may improve fertility in cows, particularly when cows are treated with shorter protocols.

The Problem of Dynamic Process Manifestation in Identification of Organic Compounds by NMR Spectroscopy

The number, shape and position of NMR spectral lines depend on dynamic processes, and this creates certain difficulties in identification of pharmaceutical substances by NMR spectroscopy. The aim of the paper was to study instances of manifestation of intramolecular dynamic processes that affect identification of organic compounds by NMR, and to illustrate the potential of the methods used for their reduction, as well as associated problems.Materials and methods: 1H and 13C spectra of the following pharmaceutical substances: «buserelin acetate», «valsartan», «goserelin acetate», «iopromide», «clopidogrel hydrogensulfate», «omeprazole», «proroxan», «risperidone», «triptorelin acetate», and «enalapril maleate» were used to demonstrate negative effects of dynamic processes. The spatial structures of conformers were established by 1H-1H ROESY experiments. The quantum-chemical calculation of geometric and thermodynamic characteristics of different conformers was carried out by the PM3 method, and electronic characteristics—by the AM1 method with the help of the HyperChem software.Results: the authors analysed intramolecular dynamic processes which are most commonly encountered in expert work: pyramidal inversion of nitrogen in a heterocyclic compound (risperidone, proroxan, clopidogrel), rotation of molecular fragments around the amide bond (valsartan, iopromide, enalapril), prototropic rearrangements (buserelin, goserelin, omeprazole, triptorelin). The change in exchange rates was explained from the perspective of the change in the system of intra- and intermolecular nonvalent interactions.Conclusions: the use of traditional methods for increasing the rate of dynamic processes (increasing the temperature and changing the solvent) does not always eliminate the negative effects of intramolecular transformations. Methods of smoothing the spectral manifestations of dynamic processes have limited application due to strong intramolecular nonvalent interactions which prevent the conversion of the dynamic process rate into fast exchange. Experts and manufacturers should take into account the manifestation of dynamic processes during identification of pharmaceutical substances by NMR spectroscopy.

Single injection of triptorelin or buserelin acetate in saline solution induces ovulation in mares the same as a single injection of hCG.

The aim of this study was to assess the efficacy of different doses of buserelin acetate and another GnRH agonist, triptorelin acetate, in saline solution in a single subcutaneous injection, to induce ovulation of growing pre-ovulatory follicle in mare and compare it with the classical treatment of a single injection of hCG. The study is split into 3 experiments over different breeding seasons in the same stud with a random distribution of treatment. The first one was to compare the injection of 6mg of buserelin with 1,500IU of hCG; the second one consisted of comparing different doses of buserelin (6mg and 3mg); and the third one compared three different doses of buserelin (3, 2 and 1mg), 0.1mg of triptorelin with 1,500IU of hCG as a control group. The results of all experiments showed the same efficacy between all treatments with mares ovulating between 24 and 48hr after injection: experiment 1: hCG (78% n=41) and buserelin 6mg (90% n=50); experiment 2: buserelin 6mg (78,1% n=192) and buserelin 3mg (78% n=341); and experiment 3: hCG (87% n=106), buserelin 3mg (84,7% n=137), buserelin 2mg (82,7% n=104), buserelin 1mg (87% n=54) and triptorelin 0.1mg (84,7% n=72). In conclusion, this study contributes to erasing the dogma that has been established since 1975 that a single injection in solution without any long-acting excipient of a GnRH agonist cannot induce ovulation in the mare. This study also shows that a injection of 0.1mg of triptorelin in solution is a good alternative for ovulation induction and is comparable to small doses of buserelin acetate in solution (1mg) and 1,500IU of the gold standard trigger hCG, mainly in countries where human formulation of buserelin is not available.

Factors associated with pregnancy rate in fixed-time embryo transfer in cattle under humid-tropical conditions of México.

The objective was to determine the effect of some factors on pregnancy rate of fixed-time embryo transfer (FTET), in cows and heifers kept under Mexican tropical conditions. Recipients females (n=405) grazing in pastures were selected according to breed group (Zebu and crosses), parity (nulliparous and multiparous), body condition score (BCS) and the presence of a corpus luteum (CL). The females were synchronized on day 0 using a progesterone vaginal device and 2 mg estradiol benzoate (EB), two groups were established. Group 1 (conventional protocol) were animals in which the progesterone device was removed on day 7. At this time, also received an injection of 50 mg cloprostenol sodium and 1 mg estradiol cypionate. Animals also received 300 IU (heifers) or 360 IU (cows) of eCG. Group 2 (J-Synch protocol) were animals in which the progesterone device was removed on day 6. Cloprotenol and eCG injections were applied as in Group 1. Additionally, on day 9, animals of group 2 received 0.01 mg buserelin acetate. Embryo transfer of in vivo or in vitro was done on day 16 and pregnancy diagnosis was realized by ultrasonography on days 23 and 53 after FTET. Statistical analyses were carried out using Chi-square tests and logistic regression. Pregnancy rate varied between farms (P<0.05). The highest pregnancy rate was for multiparous cows (66%). The recipient utilization rate was better in the J-Synch protocol (85%), and in vivo embryos (75%) had higher pregnancy rate. The diameter of the follicle and the CL had no effect on pregnancy rate (P>0.05). However, the logistic regression determined that the only significant factor on pregnancy rate was the type of embryo. In conclusion, pregnancy rate in FTET females was higher for in vivo embryos than for in vitro embryos in cows evaluated under humid tropical conditions in Mexico.

Artificial insemination with frozen semen in rabbits in Brazil: first litter reported

Brazilian rabbit breeding plays an important social role through family farming. However, one of the challenges faced is the need for new genetic material from genetically improved rabbits. The artificial insemination (AI) technique in Brazil is restricted to the use of fresh or chilled semen. Hence, this is the first report of the use of frozen semen and live kits in Brazil and probably in Latin America. The aims were to evaluate the commercial viability of frozen rabbit semen in Brazil, ranging from cryopreservation to insemination. Three bucks and three rabbit does of Flemish giant rabbits were used. Semen was collected, evaluated and diluted for freezing with CUNIFreeze. Semen was frozen in a 0.5 mL straw, thawed and then filled in a blue curve rabbit AI sheath with the doe in dorsal position with ovulation induced at the same time with buserelin acetate. The ejaculate volume was 0.8 ± 0.8 mL; the mass motility was 3.3 ± 0.6; and the total motility and vigor after dilution were 65% ± 5 points and 3.7 ± 0.6, respectively. Post-thawed, the motility and vigor were 20% ± 10 and 3,0 ± 0,0, respectively. The pregnancy rate was 66.6% with a litter sizes of 5 and 2 kits. Even with a lower result of litter size compared with refrigerated semen, this report brings a new scenario for rabbit breeding in Brazil, showing that frozen semen is a feasible technique for biobanking and mainly bringing the possibility of imported semen from genetically improved bucks solving inbreeding problems.

The effectiveness of Teucrium chamaedrys L. extracts on endometriotic implant regression in rat endometriosis model.

The aim of the present study was to investigate the therapeutic effects of Teucrium chamaedrys L. (Lamiaceae) in the experimentally induced endometriosis in rats. Endometrial tissue was implanted into the abdominal wall of thirty Sprague Dawley rats; the rats with endometriosis were randomized into five groups and treatment procedure was performed for three weeks. The treatment groups were orally treated with three different extracts of Teucrium chamaedrys. Buserelin acetate (20.00 mg) was given as a reference drug. Vehicle was administered alone to the control group. All rats were sacrified at the end of the experiment. The endometriotic implants were measured, intra-abdominal adhesions were scored and the tissue samples were histopathologically investigated. After the treatment procedure, the volumes of endometrial implant and adhesions were detected to be significantly decreased in the T. chamaedrys extracts treated groups compared to the control group. Therapeutic effect of the T. chamaedrys extracts could be attributed to the both nonpolar and polar secondary metabolites. The study conceived that the different polarity extracts of T. chamaedrys could be beneficial in the treatment of endometriosis.

Buserelin Acetate Reduces Mortality and DNA Defragmentation of Bovine Sperm Cells Exposed to Oxidative Stress

Buserelin Acetate Reduces Mortality and DNA Defragmentation of Bovine Sperm Cells Exposed to Oxidative Stress

108 Use of seminal plasma as ovulation inductor in alpacas (Vicugna pacos) embryo recipient and its effect on pregnancy rate

Alpacas, like other camelids, are classified as induced ovulators because an external stimulus is required for the occurrence of ovulation. Recent studies have identified the β-nerve growth factor (β-NGF), a protein present in the seminal plasma (SP), as responsible for inducing ovulation in this species and having potent luteotropic function. We speculate that SP can be used in reproductive biotechnologies, such as embryo transfer (ET), to increase the number of genetically superior animals produced in breeding programs. The objective of this study was to evaluate the effect of inducing ovulation with SP or a gonadotrophin-releasing hormone (GnRH) analogue on pregnancy rate in recipients of an alpaca ET program. Semen from 5 adult male alpacas was collected with an artificial vagina and diluted 1:1 (v/v) with phosphate-buffered saline (PBS; Gibco-BRL). The diluted semen was centrifuged for 30min at 1200g, and the supernatant or SP was decanted and examined for absence of sperm. Then, the SP was centrifuged again for 20min at 1200×g. The SP was stored at −80°C until the use. Female alpacas (n=38; 6-8 years) with a body condition score of 2.5-3.5 (scale 1-5) were used for the experiment. Animals were evaluated daily by transrectal ultrasonography to determine the presence of a dominant follicle ≥7mm and randomly assigned to one of two groups: (1) GnRH (n=20), 0.04mg of Buserelin acetate IM, or (2) SP (n=18): 1.0mL of seminal plasma IM. Seven days after GnRH or SP treatment (Day 0) the recipients received a fresh embryo. Alpaca donors (n=18) were given GnRH (0.04mg IM) and treated 36h later with 700IU of equine chorionic gonadotropin (ECG). Donors were mated with fertile males 5 days after ECG (Day 0 of recipients), and embryos were recovered 7 days later. Embryos of similar quality were assigned to both groups and transferred nonsurgically to the uterine horn ipsilateral to the corpus luteum. Ultrasonography examinations were performed on Day 2 to confirm ovulation and Day 25 to determine pregnancy in all of the recipients. Data was analysed by chi-squared test. Ovulation rate was not different between groups (100% each). Pregnancy rate was 45% (9 out of 20) and 44% (8 of 18) in GnRH and SP groups, respectively (P=0.77). In conclusion, SP was effective to induce ovulation in alpacas and was able to produce a pregnancy rate similar to that of GnRH as an ovulation-inducing treatment. Thus, SP can be used as an alternative for ET programs in alpacas. Research was funded by the project Role of Seminal Plasma in Reproductive Physiology and Application of Biotechnologies in Camelids (149-2017-CIENCIACTIVA).

Administration of PGF2α during the periovulatory period increased fertilization rate in superovulated buffaloes

The aim of the present study was to determine the recovery of embryonic structures (ova/embryos) and fertilization rate in superovulated buffaloes treated with PGF2α during the periovulatory period. On day 0 (D0), buffaloes at random stages of the estrous cycle were treated with an intravaginal progesterone device (P4; 1.0 g) and estradiol benzoate (EB, 2.0 mg i.m.). From D4 to D7, all buffaloes received i.m. FSH (200 mg total) twice-daily over 4 days in decreasing doses. On D6 and D7, the animals were given PGF2α analogue (0.53 mg i.m. sodium cloprostenol) and the P4 device was removed on D7. On D8, all buffaloes received GnRH (20 μg i.m. buserelin acetate). Buffaloes were then randomly allocated to one of three groups: control (Group C, n = 18), no further treatment; PGF2α analogue injection (Group IM-PGF; n = 18), four injections (0.53 mg i.m. sodium cloprostenol) 12 h apart, from D8 to D10; PGF2α analogue osmotic pump (Group OP-PGF; n = 18), s.c. osmotic mini-pump (2.12 mg sodium cloprostenol) from D8 to D10. The study had a crossover design (three treatments x three replicates). All animals underwent timed AI, 12 and 24 h after treatment with GnRH. Embryonic structures were recovered on D14. Ovarian ultrasonography was used on D8 and D14 to record follicular superstimulation and superovulatory responses. Blood samples were obtained on Days 7, 8, 9 and 10 to measure circulating concentrations of P4, E2 and PGFM. Data were analyzed by GLIMMIX procedure of SAS®. There was no effect (P = 0.58) of treatment on the total number of embryonic structures (Group C, 2.1 ± 0.8; Group IM-PGF, 2.1 ± 0.6; Group OP-PGF, 1.4 ± 0.4). There was also no effect (P = 0.93) of treatment on the recovery rate of embryonic structures (oocytes and embryos D14/CL D14). The fertilization rate was higher (P = 0.04) in Groups IM-PGF (84.6%) and OP-PGF (88.0%), which did not differ, than Group C (63.2%). The viable embryos rate was greater (P < 0.01) for Groups IM-PGF (82.0%) and OP-PGF (88.0%) than Group C (52.6%). There was no interaction between treatment and time and treatment effects for P4, E2 and PGFM concentrations. The findings showed that treatment with PGF2α during the periovulatory period has potential to increase fertilization rate and embryo production in superovulated buffaloes.

Lame cows have a subdued hypothalamus-pituitary axis as revealed by LH release in response to exogenous endocrine challenges

Lameness is a serious concern in cows due to its multilevel effects and increasing incidences. The ability of anterior pituitary and hypothalamus to release LH, a prime reproductive hormone, is unknown in lame cows. Our study tested this ability by exogenous endocrine challenge using Buserelin acetate (GnRH analogue) and estradiol valerate (EV). The study included moderately lame (L; n=7) either cyclic (LC; n=3) or anestrus (LA; n=4); and normal cyclic, non–lame (NL; n=5) cows. Progesterone blocks LH release, so it was lowered by injecting PG in cyclic cows. After 14 h, all cows were given a walk for 5 min and immediately thereafter injected with 0.008 mg GnRH and 4 h later with 1 mg of EV. Blood plasma progesterone, cortisol and LH were quantified, periodically. There was a significant delay in the onset (105.0 vs 75.0±7.0 vs 54.0±9.0 min) and duration (22.5±5.3 vs 80.0±10.8 vs 117.0±2.7 min) of LH surge in LA vs LC vs NL cows; the surge was absent in two LA cows. The altered LH profile was attributed to a 3 fold (in L) vs 1.6 fold (in NL) rise in cortisol. LH released (ng/ml) in response to EV was also low in LA (31.5±2.4) and LC (69.9±7.2) than NL (79.2±7.0) cows. In conclusion, lame cows subjected to walk exhibited reduced pituitary response to release LH following exogenous GnRH as also a reduced ability of hypothalamus to stimulate LH release in response to exogenous estradiol.

Hot-Melt Preparation of a Non-Biodegradable Peptide Implant: A Proof of Principle.

Both biodegradable and non-biodegradable peptide-loaded implants are already developed for the long-term treatment of patients, thereby reducing the frequency of drug administration. To further improve peptide formulation, extending the scope of implant-based drug delivery systems towards other polymers and processing techniques is highly interesting. In this study, as a proof-of-principle, the feasibility of hot-melt processing of a peptide active pharmaceutical ingredient was assessed by developing a non-biodegradable poly(ethylenevinyl acetate) (33% VA) implant loaded with 20% (w/w) buserelin acetate. Cross-sectional implant characterization was performed by Raman microscopy. The stability of buserelin acetate in the polymeric matrix was evaluated for 3 months under ICH stability conditions and the quantity as well as the degradation products analyzed using LC-UV methods. An in vitro dissolution study was performed as well and buserelin acetate and its degradants analyzed using the same chromatographic methods. No significant quantities of buserelin acetate-related degradation products were formed during the hot-melt preparation as well as during the stability study. Together with the consistent buserelin acetate assay values over time, chemical peptide stability was thus demonstrated. The in vitro buserelin acetate release from the implant was found to be diffusion-controlled after an initial burst release, with stable release profiles in the stability study, demonstrating the functional stability of the peptide implant. These results indicate the feasibility of preparing non-biodegradable peptide-loaded implants using the hot-melt production method and may act as a proof of principle concept for further innovation in peptide medicinal formulations.

Evaluation of uterine receptivity after gonadotropin releasing hormone agonist administration as an oocyte maturation trigger: a rodent model

In natural cycle or minimal stimulation cycle IVF, buserelin acetate (buserelin), a gonadotropin-releasing hormone agonist, is often used as a maturation trigger; however, its effect on pregnancy outcomes remains unclear. Therefore, in the present study, we compared uterine receptivity in buserelin-administered mice with that in human chorionic gonadotropin (hCG)-administered mice during the peri-implantation period. Implantation, decidualisation, and term-pregnancy were impaired following hCG, but not buserelin administration. hCG stimulated the synthesis and secretion of progesterone and oestradiol, whereas ovarian steroidogenesis in the buserelin-treated group was comparable with that in the control group. Furthermore, similar to the observation in controls, the buserelin-treated group exhibited activation of progesterone receptor signalling and inhibition of oestrogen receptor signalling in the endometrial epithelium on the day of implantation. However, epithelial progesterone signalling was not detected, and a high expression of genes downstream to oestrogen was observed on day 4 following hCG administration. These results suggest that buserelin administration does not impact uterine receptivity as it did not affect ovarian steroidogenesis and endometrial steroid signalling. Therefore, buserelin is preferred as an oocyte maturation trigger to optimise uterine receptivity during treatments involving timed intercourse, intrauterine insemination, or fresh embryo transfer following in vitro fertilisation.

Expression of prostaglandin (PG) D synthase lipocalin and hematopoietic type and PG D receptor during restart of spermatogenesis following downregulation using a slow release GnRH agonist implant in the dog.

Prostaglandin D and the associated prostaglandin D synthases (PGDS) and receptor (DP) are considered to be involved in spermatogenesis. However, the interplay of the PGDS-DP system in male reproduction is far from being understood. The expression of PGDS lipocalin (L) and hematopoietic (H) type and DP was studied in the GnRH agonist-downregulated canine testis (week, w 0) and during recrudescence of spermatogenesis after implant removal (w 3, 6, 9, 12). H-PGDS, L-PGDS and DP were present in the adult (CG), juvenile (JG) and downregulated canine testis at the mRNA level. PGDS immunohistochemistry revealed positive staining in the cytoplasm of Leydig cells (LCs) of all samples i.e., no difference between groups. mRNA expression (ratio) of L-, H-PGDS and DP did not differ between groups w 0-12 and CG. In contrast, significant differences were found for L-PGDS (p = 0.0388), H-PGDS (p < 0.001) and DP (p < 0.001) for the groups at downregulation (w0, suprelorin group, SG, profact group, PRG) compared with the control groups (JG, CG). L-PGDS expression was lowest in JG, whereas H-PGDS was significantly lower in CG compared with JG and at downregulation (p < 0.001 to p < 0.01). The highest ratio for H-PGDS and DP was observed in the dogs treated with buserelin acetate (PRG). Our data show that the PGDS-DP system is expressed in juvenile and adult canine testes and that downregulation of the testicular endocrine and germinative function significantly affects H-PGDS, L-PGDS and DP mRNA expression indicating a role in the regulation of spermatogenesis.

Ultrasound Imaging of the Testes and Accessory Sex Glands in Male Goat Treated with GnRH During Non-Breeding Season

The aim of the present study was to determine the effect of GnRH on the measurements of testes, accessory sex glands and testosterone hormonal concentrations during the non-breeding season. Eight mature healthy Egyptian Baladi male goat injected with Buserelin acetate (GnRH analogue) 8μg once weekly for one month. Ultrasonographic measurements of the scrotum and its contents and all accessory sex gland and blood samples collection were done one week before experiment and one week after the end of experiment. Results revealed that scrotal circumference, length and breadth of both testes did not differ significantly (p>0.05). While, the length and breadth of the tail of epididymis was increased significantly after treatment (p<0.05). The length and breadth of vesicular gland and bulbourethral gland differed significantly (p<0.05) after treatments. The ampulla and pars disseminata of prostate breadth was increased significantly (p<0.05). Treated bucks showed higher values for serum testosterone after treatment. In conclusion, the present results demonstrated that GnRH injection ameliorates male goat reproductive efficiency. Moreover, ultrasonography is an affirmative indicator to the response of male reproductive system to exogenous GnRH analogue treatment during the non-breeding season.

Effect of prostaglandin F2α and GnRH administration at the time of artificial insemination on reproductive performance of dairy cows.

The present study aimed to determine the effect of administrating prostaglandin F2α (PGF2α) and GnRH at the time of artificial insemination (AI) on the pregnancy per artificial insemination (P/AI) and the pregnancy survival rate of dairy cows. A number of 830 lactating Holstein cows were randomly divided into four groups. Cows in group 1 (n=200) treated with 150 µg d-cloprostenol. In group 2 (n=212), cows received 10 µg buserelin acetate, and group 3 (n=205) was treated with both 150 µg d-cloprostenol and 10 µg buserelin acetate. In addition, 213 cows were assigned as control group which received normal saline as placebo (group 4). To measure progesterone, milk samples were collected at the insemination day and five days later. Pregnancy diagnosis was performed 28 and 60 days after the insemination, and the size and number of corpus luteum (CL) and twin pregnancies were recorded. Hormone therapies had no effect on the P/AI, pregnancy survival rate, and the size and number of CL. The P/AI ratio in groups 1, 2, 3 and 4 were 38.50%, 42.92%, 41.46% and 40.84%, and the pregnancy survival rates in groups 1, 2, 3 and 4 were 84.42%, 86.81%, 88.23% and 83.91%, respectively. The probability of a twin pregnancy was significantly higher in group 1 (15.58%) than other groups. There was no significant difference between groups in terms of the offspring gender. In conclusion, the administration of d-cloprostenol or buserelin acetate at the time of AI had no effect on P/AI and pregnancy survival rate in dairy cattle under no heat stress condition, while the administration of d-cloprostenol increased the probability of twin pregnancies.

Effect of addition of buserelin acetate to the extender on motility and viability of bovine spermatozoa

The present study was aimed to determine the effect of GnRH analog (buserelin acetate) on the quality of bovine spermatozoa stored at 16° C for 24 h. Semen collected in the summer season from June to September from healthy Polish Holstein–Friesian bulls. Ejaculates were centrifuged, divided and diluted to the final concentration of 240 × 106 spermatozoa/mL using animal protein–free commercial BIOXcell® extender (IMV Technologies, L’aigle, France) (Control) or with BIOXcell® extender supplemented with buserelin acetate and stored 0, 8 and 24 h. Sperm motility parameters analysis was performed using a computer-assisted sperm analysis (CASA) system. The viability of spermatozoa was performed using flow cytometer. The addition of buserelin acetate to BIOXcell® extender did positively affect the total motility (was higher in the observed samples with the addition of 2 µg/mL and 4 µg/mL than in the control group), progressive motile (forward progressing sperm was significantly increased (p < 0.05) over the control group at the 0 h and 8 h of incubation following the supplementation of 2, 4 and 8 μg/mL buserelin acetate) and viability of spermatozoa (the number of live spermatozoa was significantly higher (p < 0.05) in 2 µg/mL and 4 µg/mL samples with buserelin acetate at 8th hour of incubation and in sample with 4 µg/mL at 24th hour of incubation compared to the control group). We recommend adding 4 µg/mL to the extender to improve the quality of bovine semen.

Evaluation of embryo collection and transfer days on the pregnancy rate of Mangalarga Marchador mares during the breeding season

Background: Proper timing for embryo collection and transfer in horses -which is critical for the success of this biotechnology- is still debated. Additionally, there is little information on this technology under tropical conditions. Objective: To determine the best day for collection and transfer of embryos in Mangalarga Marchador mares under Brazilian northeast’s conditions. Methods: Donors (n= 30) and recipients (n= 76) in diestrus phase were selected based on both clinical and gynecology examinations. Estrus was induced on both donor and recipient mares by intramuscular injection of 5 mg Dinoprost, aiming to obtain an ovulation interval of -1 to +3 between recipient and donor. Ovulation was induced with buserelin acetate when the largest follicle reached at least 35 mm in diameter. At this time, mares were subjected to artificial insemination at 48-hour intervals until ovulation. The embryos were collected on days 7, 8, and 9 after ovulation. Results: The embryo collection on day 8 was more efficient (p 0.05) than day 9, which presented the same efficiency (p>0.05) as day 7. From a total of 76 embryos transferred to the recipients, that were between days 4 and 9 after ovulation, there was no influence (p>0.05) of the day of transfer on pregnancy rate. Conclusions: The embryo collection must be performed on day 8 after ovulation, and transfer can be performed on any day of that interval (4-9) without affecting the pregnancy rate.

Serum Macro-Minerals Profile and Conception Rate following Heatsynch and Heatsynch + PRID Protocols in Subestrus Surti Buffaloes

A study was carried out to interpret the first and second service conception rate along with serum macro-minerals profile for Heatsynch (T1) and Heatsynch + PRID (T2) protocols with fix timed AI in subestrus Surti buffaloes (n=6 each), keeping one group as untreated control (T3). In Heatsynch protocol, buffaloes were administered i.m. with Inj. Busereline acetate 10 μg on day 0, Inj. Cloprostenol sodium 500 μg on day 7 and Inj. oestradiol benzoate (EB) 0.5 mg on day 8 followed by FTAIs twice on day 10, while in Heatsynch + PRID protocol the buffaloes were inserted with PRID (0.96 g of progesterone) intravaginally for 7 days together with above Heatsynch protocol. Blood samples were collected aseptically in vaccutainers from all these animals on day 0 (prior to treatment), day 4 (during treatment), day 8 (after PGF2α inj.), day of induced estrus/FTAI and on day 28 (post-treatment, day 18 post-AI), and serum samples were stored at –20°C until analyzed. The buffaloes that became pregnant at 1st service (FTAI) in the treatment group T1 and T2 were 50.00 (3/6) and 66.66 (4/6) % and at 2nd cycle 100.00 (3/3) and 50.00 (1/2) %, respectively. The corresponding conception rates for control group T3 were 33.33 (2/6) and 50.00 (2/4) %. The overall pooled CRs of 2 cycles achieved in groups T1, T2 and T3 were 100.00 (6/6), 83.33 (5/6) and 66.66 (4/6) %, respectively. The overall mean serum calcium, phosphorus and magnesium values did not differ significantly (p&gt;0.05) among T1, T2 and T3 groups at any day or within group between different sampling intervals. Heatsynch alone was better and economic over other groups in managing subestrus buffaloes.

112 Comparative study between slow freezing and vitrification on the survival rate of cryopreserved alpaca embryos post-transfer

The objective of this study was to compare the effectiveness of cryopreserving in vivo-produced alpaca embryos by slow freezing v. vitrification. The embryos were produced from 9 female alpacas at Fundo Mallkini, Puno, Peru, located at 4300m elevation. The donor alpacas were synchronized by induction to ovulate with an injection of gonadotropin-releasing hormone (0.0084mg of buserelin acetate) and natural mating with vasectomized males to male receptive donors (day of ovulation induction was considered Day 0). On Day 2, the donors were injected 700IU of eCG. On Day 7, the donors received an injection of prostaglandin F2α (0.25mg of cloprostenol) and were mated on Day 8 by fertile males (2 matings 12h apart: 0600 and 1800h). The embryos were collected at 5.5 days after fertile mating and were graded as per IETS recommendations; most of the embryos were already expanded and hatched blastocysts. Embryos were washed and maintained in holding medium (1L PBS+1g Glucose+36mg sodium pyruvate+0.4% BSA+50mg kanamycin monosulfate) at 23°C for up to 1h and distributed into 2 groups for either slow freezing for direct transfer (n=14 embryos) or vitrification (n=10 embryos). Slow freezing consisted of transfer into freezing medium (9mL of 1.5M ethylene glycol+1mL of 1.0M sucrose prepared in holding media) at 23°C, placing in 0.25-mL straws and subjected to freezing at a rate of −0.5°C/ minute to −35°C and then plunging into LN. Vitrification followed a procedure described for camel embryos whereby embryos were exposed to solutions containing increasing amounts of glycerol and ethylene glycol for fixed periods and were then loaded into an open pull straw and plunged directly into LN for storage. The cryopreserved embryos were transferred into adult alpacas at the Community of Suitucancha, Junin, Peru (1500km from the farm where the embryos were collected and cryopreserved, 4200m elevation). Embryos in the slow-freezing group were thawed in warm water at 37°C for 30s and loaded directly into the embryo transfer gun for direct transfer into 7 alpaca recipients (2 embryos per recipient). Vitrified embryos were warmed by removing the open pull straw from the LN and transferring the embryos to 2 warming solutions at 37°C with decreasing levels of vitricants and containing 0.5M galactose with a final incubation at room temperature in holding media and then transferred into 5 alpaca recipients (2 embryos per recipient). The embryos were transferred into synchronized recipients by transcervical nonsurgical method. Pregnancy diagnosis was made by transrectal ultrasound examination at 45 days post-transfer. The pregnancy rates in the slow-freezing and vitrification groups, respectively, were 2/7 (29%) and 0/5 (0%); the difference was not significant (P&amp;gt;0.05) based on Fisher’s exact test. Twin pregnancies were not detected. We consider the result with slow freezing very promising, as in previous trials we had less than 18% pregnancies. More trials with larger number of embryos per cryopreservation method are being programmed.

Daily administration of a GnRH analogue enhances sperm quality in bucks during the non-breeding season

The aim of this study was to determine if daily administration of a GnRH analogue (buserelin acetate) to bucks during the non-breeding season increases testosterone concentration and improves sperm quality. Five bucks received a daily dose of buserelin for 10 days, starting on Day 0 (first administration), and another five bucks remained as controls. Testosterone concentrations were greater in treated than in control bucks during the first hours after buserelin administration (P = 0.05), but greater in controls 10 h later (P < 0.01). Sperm mass motility and percentage of motile sperm were greater in treated (3.9 ± 0.6 and 70.1 ± 7.9%, respectively) than in control bucks (1.0 ± 0.6, P < 0.01; 45.0 ± 7.9%, P < 0.05 respectively) on Day 4. Percentage of sperm with normal morphology tended to be greater in treated than in control bucks (81.8 ± 6.2% compared with 63.5 ± 6.4% respectively, P = 0.08). The treatment decreased the percentage of sperm with mid piece defect and with bent tail (7.0 ± 1.5% compared with 12.0 ± 1.5%; 8.0 ± 1.7% compared with 13.5 ± 1.7%, treated and control bucks, respectively, P = 0.05 for both). The square root percentage of sperm with loose but heads with normal structures tended to be less in treated than control bucks (1.3 ± 0.3% compared with 0.4 ± 0.3% respectively, P = 0.06). It was concluded that daily administration of buserelin during the non-breeding season led to a rapid increase in testosterone concentration and improved sperm quality.