Can in vitro embryo production be estimated from semen variables in Senepol breed by using artificial intelligence?

Thoroughly analyzing the sperm and exploring the information obtained using artificial intelligence (AI) could be the key to improving fertility estimation. Artificial neural networks have already been applied to calculate zootechnical indices in animals and predict fertility in humans. This method of estimating the results of reproductive biotechnologies, such as in vitro embryo production (IVEP) in cattle, could be valuable for livestock production. This study was developed to model IVEP estimates in Senepol animals based on various sperm attributes, through retrospective data from 290 IVEP routines performed using 38 commercial doses of semen from Senepol bulls. All sperm samples that had undergone the same procedure during sperm selection for in vitro fertilization were evaluated using a computer-assisted sperm analysis (CASA) system to define sperm subpopulations. Sperm morphology was also analyzed in a wet preparation, and the integrity of the plasma and acrosomal membranes, mitochondrial potential, oxidative status, and chromatin resistance were evaluated using flow cytometry. A previous study identified three sperm subpopulations in such samples and the information used in tandem with other sperm quality variables to perform an AI analysis. AI analysis generated models that estimated IVEP based on the season, donor, percentage of viable oocytes, and 18 other sperm predictor variables. The accuracy of the results obtained for the three best AI models for predicting the IVEP was 90.7, 75.3, and 79.6%, respectively. Therefore, applying this AI technique would enable the estimation of high or low embryo production for individual bulls based on the sperm analysis information.

Evaluation of minimally invasive estrus synchronization protocols in brown brocket deer (Subulo gouazoubira).

This study aimed to evaluate minimally invasive protocols for estrus synchronization in the brown brocket deer (Subulo gouazoubira). Females were submitted to Latin square design, in different treatments. All females received 0.25 mg of estradiol benzoate on the first day of treatment, concomitant with one of the following sources of progesterone: (1) DIP: an intravaginal progesterone releasing device for eight days, (2) MGA1x: once a day (in the morning) oral dose of 1 mg melengestrol acetate for eight days, (3) MGA2x: twice a day (morning and afternoon) oral doses of 0.5 mg of MGA for eight days, (4) P4LA: a single i.m. administration of 75 mg of long-acting progesterone (P4LA). Eight days after the beginning of each treatment, females received an i.m. administration of 265 µg of prostaglandin (PGF2α; cloprostenol). Treatment efficacy was evaluated by manifestation of behavioral estrus after treatment and concentration of fecal progesterone metabolites (FPM). The time to onset of estrus in treatment P4LA was significantly longer (180 ± 38.9 h) compared to DIP (63 ± 6.6 h), MGA1x (53 ± 14.4 h) and MGA2x (41 ± 10.1 h) (P = 0.008). According to individual baseline FPM and FPM concentration during the days after estrus, the corpus luteum formation was suggested in all females which responded to the treatments (93.75 %). Low synchrony, longer interval between PGF2α administration and onset of estrus suggest that the P4LA dose (75 mg) is too high and not effective for S. gouazoubira. DIP, MGA 1x and MGA 2x, were effective in estrus synchronization.

True vaginal prolapse associated with retroflection of the urinary bladder in a bitch.

True vaginal prolapse is rare in bitches and is most likely to occur near whelping. A 2-year-old intact female Brazilian mastiff, 39.5 kg, had a true vaginal prolapse associated with urinary bladder retroflection; the female was in estrus, and concomitantly had diarrhea for 3 d, plus vaginal hyperplasia, and ultimately a vaginal prolapse. Ultrasound examination and retrograde urethrocystography were extremely important for detection and identification of the position (retroflection) of the urinary bladder within the prolapsed vagina. These tools, therefore, are recommended for a conclusive diagnosis and surgical planning, to avoid trans- and postoperative complications, e.g., urethral damage or bladder rupture. Prompt diagnosis and surgical correction enabled a favorable prognosis and rapid postoperative recovery, avoiding complications or ultimately death of the dog.

Prolapsus vaginal vrai associé à une rétroflexion de la vessie chez une chienne. Le prolapsus vaginal vrai est rare chez les chiennes et est plus susceptible de se produire près de la mise-bas. Une femelle dogue brésilien intacte de 2 ans, pesant 39,5 kg, a eu un prolapsus vaginal vrai associé à une rétroflexion de la vessie urinaire; la femelle était en oestrus et avait simultanément de la diarrhée pendant 3 jours, plus une hyperplasie vaginale et finalement un prolapsus vaginal. L'examen échographique et l'urétrocystographie rétrograde étaient extrêmement importants pour la détection et l'identification de la position (rétroflexion) de la vessie dans le vagin faisant prolapsus. Ces outils sont donc recommandés pour un diagnostic concluant et une planification chirurgicale, afin d'éviter les complications trans- et postopératoires, par exemple des lésions urétrales ou une rupture de la vessie. Un diagnostic rapide et une correction chirurgicale ont permis un pronostic favorable et une récupération postopératoire rapide, évitant les complications ou éventuellement le décès du chien.(Traduit par Dr Serge Messier).

Detection of Trypanosoma vivax in tissues of experimentally infected goats: what is the role of adipose tissue in the life cycle of this protozoon?

Trypanosomiasis, caused by Trypanosoma vivax, is responsible for great economic losses among livestock in Africa and South America. During the life cycle of these parasites, they may present different morphological, metabolic and physiological characteristics depending on the interactions that are encountered at each point of their life cycle. Although T. vivax is frequently reported in the circulation of its mammalian hosts, it has the ability to migrate to the tissues of these individuals. However, this characteristic is poorly understood. In this context, we aimed to investigate the presence of T. vivax and the changes caused in different tissues of experimentally infected goats. Despite the animals were not perfused before tissues collection, using different approaches, we demonstrated its presence in different samples, including in the adipose tissue and skin of infected animals. In addition, a mononuclear inflammatory reaction, mostly characterized by an infiltrate of lymphocytes, plasma cells and macrophages were observed. The results highlight the possibility that, like other trypanosomatids, T. vivax may use these tissues during its life cycle. Future studies aiming to elucidate the length of time for which T. vivax remains active in these sites, and whether it uses these sites as a refuge from trypanocidal drugs, and whether it is capable of recolonizing the blood circulation, are much needed.

Silvopastoral system is an alternative to improve animal welfare and productive performance in meat production systems.

Climate change is a reality and global surface temperature is projected to rise substantially in the next 80 years. Agriculture practices will have to adapt to climate change, and also help to mitigate this effect using, among other strategies, forest conservation and management. Silvopastoral systems have been adopted in tropical climate livestock areas but their benefits on thermal comfort and reproductive performance of beef cows are not completely known. Therefore, our aims were to compare the microclimate of silvopastoral and intensive rotational unshaded grazing systems in different months and to evaluate physiological variables (Exp. 1 and 2), metabolism, and in vitro embryo production (Exp. 2) in crossbred beef females. Our hypothesis is that the silvopastoral system can improve the thermal comfort of beef heifers and cows and, consequently, also improve dry matter intake, body weight gain, and in vitro embryo production when compared to the unshaded rotational grazing system. In Exp 1, the silvopastoral system decreased body temperature and increased welfare and performance of heifers. In Exp. 2, the silvopastoral system enhanced the body weight but did not affect metabolism and the general reproductive performance, but increased the recovery rate of oocytes in primiparous cows.

Detection of Trypanosoma vivax in tissues of experimentally infected goats: what is the role of adipose tissue in the life cycle of this protozoon? / Detecção de Trypanosoma vivax em tecidos de caprinos experimentalmente infectados: qual é o papel do tecido adiposo no ciclo de vida desse protozoário?

Abstract Trypanosomiasis, caused by Trypanosoma vivax, is responsible for great economic losses among livestock in Africa and South America. During the life cycle of these parasites, they may present different morphological, metabolic and physiological characteristics depending on the interactions that are encountered at each point of their life cycle. Although T. vivax is frequently reported in the circulation of its mammalian hosts, it has the ability to migrate to the tissues of these individuals. However, this characteristic is poorly understood. In this context, we aimed to investigate the presence of T. vivax and the changes caused in different tissues of experimentally infected goats. Despite the animals were not perfused before tissues collection, using different approaches, we demonstrated its presence in different samples, including in the adipose tissue and skin of infected animals. In addition, a mononuclear inflammatory reaction, mostly characterized by an infiltrate of lymphocytes, plasma cells and macrophages were observed. The results highlight the possibility that, like other trypanosomatids, T. vivax may use these tissues during its life cycle. Future studies aiming to elucidate the length of time for which T. vivax remains active in these sites, and whether it uses these sites as a refuge from trypanocidal drugs, and whether it is capable of recolonizing the blood circulation, are much needed.

Resumo A tripanossomíase, causada por Trypanosoma vivax, é responsável por grandes perdas econômicas na bovinocultura da África e da América do Sul. Durante seu ciclo de vida, o parasita pode apresentar diferentes características morfológicas, metabólicas e fisiológicas em função das interações que ele encontra em cada ponto do seu ciclo. Embora o T. vivax seja reportado, frequentemente, na circulação dos seus hospedeiros mamíferos, o protozoário tem a capacidade de migrar para os tecidos desses indivíduos. Entretanto, essa característica é pobremente conhecida. Neste contexto, o objetivo foi verificar a presença, assim como as alterações causadas pelo T. vivax nos diferentes tecidos de caprinos experimentalmente infectados. Apesar dos animais não terem sido perfundidos antes da coleta dos tecidos, utilizando-se diferentes abordagens, foi evidenciada a presença do T. vivax em diferentes amostras teciduais, incluindo no tecido adiposo e pele dos animais infectados. Além disso, foi observada reação inflamatória mononuclear, caracterizada majoritariamente por infiltrado de linfócitos, plasmócitos e macrófagos. Os resultados evidenciam a possibilidade de que, assim como outros tripanossomatídeos, T. vivax pode usar esses tecidos durante o seu ciclo de vida. São necessários futuros estudos, objetivando elucidar o período em que o T. vivax permanece ativo nesses sítios, se ele utiliza esses locais como refúgio das drogas tripanocidas, e se ele é capaz de recolonizar a circulação sanguínea.

"Which Factors Affect Pregnancy Until Calving and Pregnancy Loss in Buffalo Recipients of in vitro Produced Embryos?"

In vitro embryo production and embryo transfer (ET) in buffaloes has been developed for decades. However, most studies are focused on the donor or laboratory improvements, and there is a lack of reports regarding the recipients. Therefore, our aim was to investigate factors associated to pregnancy (P/ET), pregnancy loss (PL), and calving rates in buffalo recipients. The studied factors were season, recipient parity, the synchronization protocol, the CL diameter, asynchrony between the embryo and the recipient, the day of the recipient estrous cycle, the embryo (fresh vs. vitrified), the day of embryo development, and the embryo stage. These retrospective data, from a program of in vitro produced embryos, were analyzed by logistic regression, and the odds ratio was also estimated. Two factors were related to P/ET and the calving rate: (1) progesterone associated to estradiol plus eCG protocol for fixed time ET tended to affect positively P/ET on day 30 (41.9 vs. 36.1%, respectively; P = 0.07; AOR = 1.28) and P/ET on day 60 (37.8 vs. 36.1%, respectively; P = 0.09; AOR = 1.08) compared to the Ovsynch protocol; and (2) the CL diameter (≥14.5 mm) at transfer increased P/ET on day 30 (47.4 vs. 32.5%; P < 0.01; AOR = 1.87) and on day 60 (45.3 vs. 27.7%; P < 0.01; AOR = 2.16), and also the calving rate (37.9 vs. 21.7%; P < 0.01; AOR = 2.20). PL was greater when ET was done in the nonbreeding season compared to the breeding season (PL 30-60: 12.8 vs. 0.0%, P = 0.01; AOR > 999.99; PL 60-calving: 26.8 vs. 3.6%, P = 0.03; AOR = 9.90; and PL 30-calving: 36.2 vs. 3.6%, P = 0.01; AOR = 15.30). In conclusion, the data of our study indicated that the synchronization protocol, the CL diameter, and ET during the breeding season impacted the reproductive efficiency of buffalo recipients.

Extracellular vesicles and its advances in female reproduction.

Intercellular communication is an essential mechanism for development and maintenance of multicellular organisms. Extracellular vesicles (EVs) were recently described as new players in the intercellular communication. EVs are double-membrane vesicles secreted by cells and are classified according to their biosynthesis, protein markers and morphology. These extracellular vesicles contain bioactive materials such as miRNA, mRNA, protein and lipids. These characteristics permit their involvement in different biological processes. Reproductive physiology is complex and involves constant communication between cells. Different laboratories have described the presence of EVs secreted by ovarian follicular cells, oviductal cells, in vitro produced embryos and by the endometrium, suggesting that EVs are involved in the development of gametes and embryos, in animals and humans. Therefore, is important to understand physiological mechanisms and contributions of EVs in female reproduction in order to develop new tools to improve in vivo reproductive events and assisted reproductive techniques (ARTs). This review will provide the current knowledge related to EVs in female reproductive tissues and their role in ARTs.

Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis.

In vitro oocyte transport through the oviduct of buffalo and crossbred beef cows / Transporte de oócitos in vitro através do oviduto de búfalas e vacas de corte cruzadas

The present study was conducted to verify if the elevation of plasma concentrations of estradiol during superovulatory treatments affects the oocyte transport in buffalo females, as well as if the inferior quality of buffalo oocytes and/or some functional difference on the oviduct of these animals is responsible for the low embryo recovery rate in superovulated buffaloes when compared to cows subjected to the same treatment. Oviducts of 10 buffaloes and 15 of cows, treated to induce a single ovulation were used. The oviducts were placed on Petri dishes and received the following treatments: 5 buffalo oocytes with no E2 (G-BufBuf and G-BovBuf), 5 bovine oocytes with no E2 (G-BufBov and G-BovBov), 5 buffalo oocytes with E2 (G-BufE2Buf and G-BovE2Buf) and 5 bovine oocytes with E2 (G-BufE2Bov and G-BovE2Bov; factorial 2x2x2). Oocytes were incubated for 24h. Subsequently, oviducts were washed and oocytes were recovered and counted. Since no interactions were found between E2 treatment, oviducts and oocytes species, main effects were analyzed separately. Recovery rate and number of oocytes was higher on cattle compared to buffaloes (35.0+8.6% and 1.4+0.3 vs. 10.0±4.6% and 0.5±0.2, respectively; p<0.05); no effect of E2 treatment was observed on recovery rate and number of oocytes (29.8±9.0% and 1.3±0.4 vs. 16.9±6.1% and 0.7±0.2, respectively; p>0.05); the number of buffaloes and bovine oocytes recovered were similar (1.4±0.4 and 0.6±0.2, respectively; p>0.05). Oocytes recovery rate showed a trend (P=0.07) to be higher when buffalo oocytes were implanted when compared to bovine oocytes (35.2±9.2% vs. 12.9±5.4%). Present results suggest that oocyte transport by the oviduct of buffaloes and bovine was not dependent on oocytes species or E2 supplementation to the culture medium.

O presente estudo foi realizado para verificar se a elevação das concentrações plasmáticas de estradiol durante os tratamentos superovulatórios afeta o transporte dos oócitos em fêmeas bubalinas, bem como se a qualidade inferior dos oócitos de búfalos e/ou alguma diferença funcional no oviduto destes animais é responsável pela baixa taxa de recuperação de embriões em búfalas superovuladas quando comparadas a vacas submetidas ao mesmo tratamento. Foram utilizados 10 ovidutos de búfalas e 15 de vacas, tratadas para a indução de ovulação única. Os ovidutos foram colocados em placas de Petri e receberam os seguintes tratamentos: sem E2 e inseridos com 5 oócitos de búfalas (G-BufBuf e G-BovBuf); sem E2 e com 5 oócitos de vacas (G-BufBov e G-BovBov); com E2 e com 5 oócitos de búfalas (G-BufE2Buf e G-BovE2Buf); e com E2 e com 5 oócitos de vacas (G-BufE2Bov e G-BovE2Bov; fatorial 2x2x2). Posteriormente, foram incubados por 24h e, após esse período, foram lavados para a recuperação e contagem dos oócitos. Como não foi verificado efeito de interação, foram analisados os efeitos principais. O número e a taxa de recuperação de oócitos foi maior em ovidutos de vacas que de búfalas (1,4±0,3/35,0±8,6% vs. 0,5±0,2/10,0±4,6%; P<0,05). Foi verificado que o tratamento com ou sem E2 não interferiu no número e na taxa de recuperação de oócitos (1,3±0,4/29,8±9,0% vs. 0,7±0,2/16,9±6,1%; P>0,05). Não foi verificada diferença no número de oócitos de búfalas ou de vacas recuperados (1,4±0,4 e 0,6±0,2; P>0,05). Observou-se também que houve tendência (P=0,07) de maior taxa de recuperação de oócitos de búfalas que de vacas (35,2±9,2% vs. 12,9±5,4%). Os dados são indicativos de que o transporte de oócitos pelo oviduto de búfalas e de vacas independe da espécie do oócito e não é influenciado pelo E2.

Xenooplasmic transfer between buffalo and bovine enables development of homoplasmic offspring.

Nuclear-mitochondrial incompatibilities may be responsible for the development failure reported in embryos and fetuses produced by interspecies somatic cell nuclear transfer (iSCNT). Herein we performed xenooplasmic transfer (XOT) by introducing 10 to 15% of buffalo ooplasm into bovine zygotes to assess its effect on the persistence of buffalo mitochondrial DNA (mtDNA). Blastocyst rates were not compromised by XOT in comparison to both in vitro fertilized embryos and embryos produced by transfer of bovine ooplasm into bovine zygotes. Moreover, offspring were born after transfer of XOT embryos to recipient cows. Buffalo mtDNA introduced in zygotes was still present at the blastocyst stage (8.3 vs. 9.3%, p = 0.11), indicating unaltered heteroplasmy during early development. Nonetheless, no vestige of buffalo mtDNA was found in offspring, indicating a drift to homoplasmy during later stages of development. In conclusion, we show that the buffalo mtDNA introduced by XOT into a bovine zygote do not compromise embryo development. On the other hand, buffalo mtDNA was not inherited by offspring indicating a possible failure in the process of interspecies mtDNA replication.

Perfil de FSH e LH na divergência folicular em novilhas Nelore (Bos indicus) / FSH and LH profiles during follicle deviation in Nelore heifers (Bos indicus)

O presente estudo teve como objetivo traçar o perfil de gonadotrofinas em 12 novilhas Nelore, a fim de testar a hipótese de que há declínio nas concentrações de FSH e elevação transitória nos níveis de LH durante a seleção folicular. A partir da ovulação (D0) foram colhidas amostras de sangue da veia jugular a cada 12 horas até o D5, para a dosagem de FSH e LH plasmáticos. Empregou-se o método de radioimunoensaio de duplo anticorpo. A sensibilidade do ensaio de LH foi de 0,02ng/ml e a de FSH de 0,005ng/ml. Os coeficientes de variação intra-ensaio foram 13,6% e 18,8%, respectivamente. Os dados (média±EPM) foram normalizados para o momento da divergência folicular e, posteriormente, analisados por ANOVA e por regressão inear, cúbica e quadrática. Também foi empregado o Teste t para comparação entre o ponto mais alto e o mais baixo da curva. Não se verificou efeito de tempo sobre as concentrações plasmáticas de FSH e de LH quando se utilizou análise de variância e de regressão. Entretanto, através do Teste t pontual, o FSH atingiu as menores concentrações plasmáticas 36 (0,40±0,05ng/ml) e 60 horas (0,42±0,04ng/ml) após a divergência, comparativamente às 36 horas anteriores ao desvio, quando as concentrações foram máximas (0,63±0,08ng/ml). Conclui-se, portanto, que há declínio nas concentrações plasmáticas de FSH, contudo, não foi comprovada elevação transitória nas concentrações de LH próximo ao momento do desvio folicular em fêmeas Nelore.

Present study aimed to evaluate gonadotropins profiles in 12 Nelore heifers, in order to test the hypothesis that FSH concentrations decrease and LH presents a transient increase during follicle selection. Blood samples from jugular vein were harvested twice daily starting at the time of ovulation (D0) until D5. Plasma samples were assayed for FSH and LH by double antibody radioimmunoassay method. LH and FSH assay sensitivity was 0,02ng/ml and 0,005ng/ml, respectively. The intraassay coefficient of variation was 13,6% and 18,8%, respectively. Data (mean±SEM) were normalized to follicle deviation and analyzed by ANOVA and by linear, cubic, and quadratic regressions. Comparisons between higher and lower FSH values were also performed by T-test. There was no effect of time in plasmatic FSH and LH circulating levels when variance analysis or regression analysis were performed. However, by T-test, FSH concentrations reached the lowest plasmatic levels 36 (0,40±0,05ng/ml) and 60 hours (0,42±0,04ng/ml) after follicular deviation, comparatively to 36 hours before deviation, when the concentrations were maximal (0,63±0,08ng/ml). In conclusion, there is a FSH decrease, although a transient LH elevation has not been confirmed encompassing follicle deviation in Nelore females.