FSH dose and strategy of administration during ovarian stimulation alter the gene expression profile in ovine cumulus-oocyte complexes.

Ovarian stimulation is an important tool to increase the number of oocytes obtained by laparoscopy for the in vitro production of embryos (IVP). In sheep, different concentrations of FSH administered in single dose (SD) or multiple doses (MD) have been adopted. In parallel, the oocyte quality is fundamental for IVP success, so strategies to produce more competent oocytes have been evaluated. The aim of this study was to evaluate the gene expression profile of BCB+ COC from different hormonal protocols of ovarian stimulation in Santa Inês ewes. To achieve that, a cross-over design was used, where 12 pluriparous ewes had their follicular wave synchronized (Balaro et al., Domest Anim Endocrinol, 54: 10-14, 2016). At 80 h after progestogen implant removal, all ewes received a new vaginal sponge and it started the stimulation by administration of: 80 (Group 1 - 80-SD) or 120 (Group 2 - 120-SD) mg FSH (Folltropin-V®, Bioniche Animal Health, Ontario, Canada) and 300 IU of eCG both in single dose, or 80 (Group 3 - 80-MD) or 120 (Group 4 - 120-MD) in decreasing doses (50/30/20%) every 12 h. The COCs were recovered by laparoscopy and classified morphologically in grade I / II (homogeneous ooplasm and more than 3 cumulus cells layers), III (homogeneous ooplasm and less than 3 cumulus cells layers or partially denuded) and IV (heterogeneous ooplasm or degenerate). For inference of the development competence GI, II and III COCs were stained with bright cresyl blue (BCB) and classified into: BCB+ (competent) and BCB- (non-competent). These variables were compared by ANOVA followed by Tukey test. The abundance of mRNA that encodes proteins associated with steroidogenesis (STAR, FSHr, LHr and ER?), oocyte quality (MATER, BMP15, GDF9 and ZAR1) and apoptosis (BAX and Bcl-2) was assessed by real-time qPCR normalized with GAPDH in BCB+ COCs. The abundance of gene transcripts associated with steroidogenesis was down-regulated (P <0.05) with increasing FSH concentration, when administration was performed in a single dose (80-SD and 120-SD). On the other hand, when the administration was performed in MD, only the LHr was down-regulated (80-MD and 120-MD). In the 80-MD group, FSHr and Er? were down-regulated (P <0.05) in comparison with 80-SD. For genes related with oocyte quality, 80-MD showed up-regulation (P <0.05) to MATER (when compared to 80-SD), ZAR1 and MATER (compared to 120-SD). Nonetheless, apoptosis genes were not affected. These data demonstrate that the FSH dose and strategy of administration affect the gene expression profile in ovine COCs. Subsequent studies are necessary to assess the effect of this change on maturation rate and developmental competence.Proceedings of the 31st Annual Meeting of the Brazilian Embryo Technology Society (SBTE), Cabo de Santo Agostinho, PE, Brazil, August 17 to 19, 2017

Mini-Percoll processing of domestic ruminant frozen-thawed semen dispenses the use of heparin in capacitating medium.

Sperm capacitation is a prerequisite for mammal successful fertilization. Although usually a capacitating substance such as heparin is used during sheep in vitro fertilization, evidences suggest that the cryopreservation process and Percoll technique could induce spontaneous capacitation. This study aimed to compare ovine, caprine and bovine frozen-thawed sperm after mini-Percoll processing on sperm parameters, receiving or not heparin supplementation. In conclusion, frozen-thawed ovine, caprine and bovine spermatozoa processed with mini-Percoll behave similarly regarding to capacitation status and does not require heparin supplementation during in vitro incubation to achieve capacitation. [Processamento pela técnica de mini-Percoll em sêmen congelado/descongelado de ruminantes domésticos dispensa o uso da heparina em meio capacitante].Edição dos anais do XXII Congresso Brasileiro de Reprodução Animal (CBRA), Santos, SP, Brasil, maio 2017

Mini-percoll technique induces Similar capacitation features in domestic ruminant frozen-thawed spermatozoa regardless of the presence of heparin.

Background: Sperm capacitation is a process consists of a series of functional, biochemical, and biophysical modifications that render the ejaculated sperm competent for oocyte fertilization. Secreted by the female reproductive tract epithelium, heparin promotes capacitation by binding to and removing seminal plasma proteins, which are adsorbed to the sperm PM and would inhibit capacitation. There is substantial evidence that cryopreservation promotes capacitation-like changes in bull, ram and buck sperm. Our general hypotheses were: (a) cryopreserved ram sperm suffer capacitation more quickly than buck and bull sperm under the same conditions; (b) the capacitation status of ruminant cryopreserved sperm is similar whether or not heparin is present after the mini-Percoll technique; and (c) ruminant frozen-thawed sperm selected by mini-Percoll and incubated within media without heparin supplementation is not impaired in terms of capacitation status and sperm agglutination. This study aimed to compare sperm parameters of ovine, caprine, and bovine frozen-thawed sperm after mini-Percoll processing followed by incubation with or without heparin supplementation. Materials, Methods & Results: Commercial semen of all species were used. Sperm samples were selected by mini-Percoll and supplemented (or not) with heparin within an incubation medium for 18 h. Sperm kinematics (CASA system analyzes), capacitation status (CTC staining) and sperm agglutination were evaluated after thawing, mini-Percoll, 1.5 h, 3 h, 6 h and 18 h. In comparison with post-thawing analysis, ovine species demonstrated a reduction (P 0.05). In caprine and bovine species, a lower (P < 0.05) rate of sperm agglutination was observed in the presence of heparin at 18 h of incubation. In the absence of heparin, ovine samples showed a higher (P < 0.05) agglutination rate compared to the bovine species after long incubation period. Discussion: The present study compared sperm parameters (sperm kinematics, agglutination rate and capacitation status) of ruminant frozen-thawed sperm after mini-Percoll selection followed by in vitro incubation with or without heparin supplementation. In this study, it was observed the same rate of capacitated cells after the sperm selection (min-Percoll) between ruminant species. This indicate that the capacitation process occurs similarly between ruminant species, refuting the first hypothesis of this study. The presence of heparin did not influence the capacitation status of ruminant frozen-thawed sperm after mini-Percoll selection, it demonstrates that the second hypothesis was supported by this study making more economic and practical the use of ruminant frozen-thawed semen. The absence of heparin in the incubation medium did not harmed the capacitation status and sperm agglutination of ruminant frozen-thawed sperm. This supported the third hypothesis of the current study and indicate that the use of mini-Percoll technique regardless the presence of heparin could be a useful alternative for the preparation of ruminant frozen-thawed sperm. In conclusion, the capacitation status of ruminant frozen-thawed sperm is similar whether or not heparin is present after the mini-Percoll technique

L-carnitine supplementation during vitrification did not improve survival and quality rates, but altered CrAT and PRDX1 expression in in vivo-produced ovine embryos.

Embryo cryodamage is observed mainly at metabolic and molecular aspects and it impairs post warming quality and survival rates. This study aimed to evaluate the effect of L-carnitine (LC) supplementation during either vitrification or post warming solutions on the 6-7th day of in vivo-produced ovine embryos. LC (3.72 mM) was added to vitrification (Experiment 1; C1: control; LC1: supplemented embryos) or warming solutions (Experiment 2; C2; LC2). In vitro culture (IVC) of warmed embryos was performed for 72 h at 38,5 °C, 5% CO2 and 5% O2 to evaluate survival rates in both Experiments. In Experiment 1, reactive oxygen species (ROS) levels were measured by CellROX Green staining, total cell number (TCN) by Hoechst 33342, number of apoptotic cells by caspase-3 immunofluorescence staining protocol, apoptotic index evaluation in both groups. Gene expression analysis of carnitine palmitoyltransferase 1 and 2 (CPT1 and CPT2), carnitine O-acyltransferase (CrAT) and peroxiredoxin 1 (PRDX1), were performed by RT-qPCR (ACTB as endogenous control) in Experiments 1 and 2 and results were compared to fresh embryos (FE). Averages of survival rates were compared by the Chi-Square test. Means of TCN, apoptotic cells, apoptotic index and fluorescence intensity were compared by Student's t-test, at 5% significance level. Survival rates were similar between groups (p> 0.05) in Experiments 1 (68.7%, C1 vs 81.8%, LC1) and 2 (48.5%, C2 vs 64.7%, LC2). In Experiment 1, ROS levels at 24 h of IVC (85.83 ± 68.37 x 1010, C1 vs 89.04 ± 84.48 x 1010, LC1), total cell number at 24 h (89 ± 22, C1 vs 82.2 ± 28, LC1) and 72 h (86 ± 19.9, C1 vs 68.5 ± 25.26, LC1), apoptotic cells (3.75 ± 1.48, C1 vs 4.50 ± 4.72, LC1) and apoptotic index (4.37 ± 1.45, C1 vs 5.23 ± 4.72, LC1) at 72 h of IVC did not differ (p> 0.05) between C1 and LC1. Gene expression analysis showed no differences in CPT1 and CPT2 mRNA relative abundance in embryos of both experiments compared to FE, however, CrAT was downregulated (p< 0.05) in C1 and PRDX1 was downregulated (p< 0.05) in both C1 and LC1, compared to FE. Moreover, CrAT and PRDX1 were upregulated (p< 0.05) in C2 and CrAT was downregulated (p< 0.05) in LC2, in relation to FE. In conclusion, although the short-term LC supplementation at 3.72 mM during cryopreservation did not improve post-warming survival and morphological parameters of the evaluated embryos, it was able to modulate expression of genes related to energy homeostasis (CrAT) and oxidative stress (PRDX1), proving to be beneficial, in both forms of supplementation, to in vivo-produced ovine embryos.".Proceedings of the 31st Annual Meeting of the Brazilian Embryo Technology Society (SBTE); Cabo de Santo Agostinho, PE, Brazil, August 17th to 19th, 2017. Abstracts

Repetibilidade de resposta para produção in vivo de embriões em ovelhas Santa Inês.

Resumo: Tem sido observado em caprinos e bovinos que animais com desempenho satisfatório em programas de superovulação tendem a manter esta boa resposta em protocolos consecutivos. Esta característica tem uma grande aplicabilidade, uma vez que um primeiro programa para produção de embriões poderia servir para triar animais com maior potencial de resposta. Assim, apenas as doadoras com histórico de alto potencial de resposta seriam incluídas em futuras superovulações, o que poderia aumentar a eficiência da MOTE. Levando em conta estes conceitos, objetivou-se verificar se borregas da raça Santa Inês mantém um padrão de resposta quando submetidas a três superovulações consecutivas para a produção in vivo de embriões. [Repeatability of response for embryo in vivo production in Santa Inês sheep].Resumo apresentado nos anais do XXII Congresso Brasileiro de Reprodução Animal, Santos, SP, 2017

Efeito de diferentes protocolos hormonais para superestimulação ovariana sobre o número e a qualidade de oócitos em ovelhas da raça Santa Inês.

Resumo: A obtenção de oócitos competentes é um dos fatores que influenciam o sucesso da produção in vitro de embriões em ovinos. Objetivou-se avaliar o efeito de protocolos hormonais na quantidade e qualidade de complexos cumulus-oócitos (CCOs) em ovelhas da raça Santa Inês. Para o número de oócitos GII, houve efeito (P0,05) na taxa de CCOs BCB+ nos diferentes tratamentos: T1(72%), T2 (88%), T3 (80%) ou T4 (76%). Assim, recomenta-se utilizar o T1, devido à praticidade de administração única ou T3, protocolo que possibilita reduzir os custos e efeitos colaterais do eCG. [Effect of different hormonal protocols for ovarian superstimulation on the number and quality of oocytes in Santa Inês ewes].Edição dos anais do XXII Congresso Brasileiro de Reprodução Animal (CBRA), Santos, SP, Brasil, maio 2017

Cervical transposition test at estrus as a tool to select ewes for transcervical embryo collection.

The use of multiple ovulation and embryo transfer (MOET) program in sheep is limited by anatomical particularities of ovine cervix, making non-surgical embryo collection method (transcervical) more difficult or even impracticable. Therefore, the cervical morphology screening prior MOET becomes an interesting strategy. Thus, this study aimed to evaluate a cervical transposition method as a tool to select ewes able of being submitted to non-surgical embryo collection. The high SENS and Acc verified in the study demonstrated that cervical transposition test at the estrus using a Hegar dilator has the potential to be included in MOET programs as a screening strategy to direct ewes for a surgical or non-surgical embryo collectio

Follicular wave synchronization on in vivo embryo production in Santa Ines sheep.

Abstract: This study aimed to evaluate two hormonal protocols for synchronization of follicular wave emergence on in vivo embryo production in Santa Ines sheep under tropical conditions. Twenty two nulliparous Santa Ines sheep, kept on intensive system, were used. Group one (GT, n = 10) received an intravaginal implant containing 0.33 g of progesterone (Eazi-Breed CIDR® Sheep and Goats, Zoetis Ltda, São Paulo, Brazil) for all period of hormonal protocol and 0.24 mg of cloprostenol (Estron®, Agener Union, São Paulo, Brazil) i.m.. Group two (GEm; n = 12) received the synchronization of the follicular wave emergence as proposed by Balaro et al. (2016). The superovulation started after 56 and 80 hours, from GT and GEm, respectively. For both groups, 200 mg of FSHp/per animal were administered (Folltropin®, National Pharmaceutical Chemistry Union S/A, São Paulo, Brazil) in six decreasing doses (25%/25%, 15%/15% and 10%/10%) every 12 hours. In GEm, at the first FSHp dose, an intravaginal sponge impregnated with 60 mg of medroxyprogesterone acetate (Progespon®, Schering Plough, São Paulo, Brazil) was inserted and at the fifth dose, ewes also received 0.24 mg of cloprostenol (Estron®, Agener Union, São Paulo, Brazil). The intravaginal implant (GT) or sponge (GEm) was taken at the last FSHp dose. Subsequently, estrus detection and mating occurred every 12 hours. Seven days after the last FSH dose, embryos were collected by surgical uterine flushing. An ultrasound (Sonoscape S6®, Sonoscape, Yizhe Building, Yuquan Road Shenzhen, China) equipment coupled to a 7.5 MHz linear transducer (transrectal) was used to assess the follicular population on the progestagen insertion (D0), in the first superovulatory dose and for CL count immediately before embryo collection. Furthermore, regardless of the occurrence of premature regression of CL (PRCL), all ewes were collected. Normal and non-normal parametric data were evaluated by t-test and MannWhitney, respectively. Frequency data were evaluated by Fisher's exact test. It was considered as significant when P 0.05) were detected, between GT and GEm, in the follicular population at the day of the progestagen insertion (7.8 ± 2.6 vs. 6.0 ± 1.6) and the first FSHp dose (8.1 ± 2.6 vs. 8.9 ± 2.3). The number of CL were also similar (P > 0.05) between G1 (6.9 ± 5.1) and G2 (7.1 ± 3.1). The number of animals with PRCL in GT was 60% (6/10), greater than the 8.3% (1/12) found in GEm (P 0,05) entre GT (6,9 ± 5,1) e GEm (7,1 ± 3,1). O número de animais com RPCL no GT foi de 60% (6/10), superior (P<0,01) aos 8,3% (1/12) encontrados no GEm. O número de estruturas coletadas (6,6 ± 4,3 vs. 0,6 ± 0,7; P<0,01) e embriões viáveis (4,6 ± 3,9 vs. 0,3 ± 0,5; P<0,01) foram superiores no GEm comparado ao GT. A taxa de recuperação também diferiu entre os grupos experimentais, sendo superior no GEm (75,6% vs. 8,1%; P<0,01). A maior taxa de RPCL no GT contribuiu para o menor número de embriões viáveis obtidos. Assim, indica-se o GEm ao objetivar a produção de embriões em ovinos da raça Santa Inês manejados sob condições tropicais.Proceedings of the 30th Annual Meeting of the Brazilian Embryo Technology Society (SBTE); Foz do Iguaçu, PR, Brazil, August 25th to 27th, 2016, and 32nd Meeting of the European Embryo Transfer Association (AETE); Barcelona, Spain, September 9th and 10th, 2016