The waveless model: clinical and research applications of follicular wave suppression in cattle / Modelo waveless: aplicações clínicas e na pesquisa da supressão das ondas foliculares em bovinos

The follicular growth waves are directly linked to the fluctuations in plasma gonadotrophins, which are controlled by the hypothalamic GnRH release pattern. Therefore, if the actions of the GnRH are inhibited or blocked, the final stages of the antral follicle growth are suppressed, resulting in an induced anestrus (a.k.a. waveless model). In the human medicine, GnRH agonists or antagonists are broadly used in the control of ovarian disfunctions, as well as in the preparation of women for assisted reproductive cycles. In cattle, a similar effect can be obtained by active immunization against GnRH. This was shown to be a viable strategy, for example, for the control of chronic cases of cystic ovarian disease in oocyte donors. However, on shall take into account the substantial individual variation on the immune response and, consequently, the lack of control of the duration of the anestrus induced. The waveless model is also very useful as a research model, once it controls the potential interference of the endogenous FSH and LH, improving the sensitivity of essays with exogenous hormones and consequently reducing the required number of replicas within studies.(AU)

O padrão de crescimento folicular em ondas está diretamente associado às flutuações nas concentrações plasmáticas de gonadotrofinas, controladas por sua vez pelo padrão de liberação de GnRH hipotalâmico. Desta forma, a inibição ou bloqueio da ação do GnRH suprime as etapas finais do crescimento folicular, resultando em anestro induzido (também chamado modelo waveless). Na medicina humana, agonistas ou antagonistas de GnRH são utilizados tanto no controle de disfunções ovarianas quanto na preparação de pacientes para procedimentos de reprodução assistida. Em bovinos, este efeito pode ser obtido pela imunização ativa contra GnRH, e mostrou-se estratégia viável, por exemplo, no controle de casos crônicos de doença ovariana cística em doadoras de oócitos. Contudo, é importante considerar a grande variação individual na resposta à imunização e consequente impossibilidade de controlar a duração do anestro induzido. O modelo waveless também é de grande utilidade na pesquisa, uma vez que elimina a potencial interferência do FSH e LH endógenos, aumentando a sensibilidade nos ensaios com hormônios exógenos e consequentemente reduzindo o número de réplicas necessárias nos estudos.(AU)

Use of new recombinant proteins for ovarian stimulation in ruminants

Currently, gonadotropin products (follicle stimulating hormone, FSH, and luteinizing hormone, LH) used in animal reproduction are produced by extraction and purification from abattoir-derived pituitary glands. This method, relying on animal-derived materials, carries the potential risk of hormone contamination and pathogen transmission. Additionally, chorionic gonadotropins are extracted from the blood of pregnant mares (equine chorionic gonadotropin; eCG) or the urine of pregnant women (human chorionic gonadotropin; hCG). However, recent advancements have introduced recombinant gonadotropins for assisted animal reproduction therapies. The traditional use of FSH for superovulation has limitations, including labor requirements and variability in superovulation response, affecting the success of in vivo (SOV) and in vitro (OPU/IVEP) embryo production. FSH treatment for superstimulation before OPU can promote the growth of a homogenous follicular population and the recovery of competent oocytes suitable for IVEP procedures. At present, a single injection of a preparation of long-acting bovine recombinant FSH (rFSH) produced similar superovulation responses resulting in the production of good-quality in vivo and in vitro embryos. Furthermore, the treatment with eCG at FTAI protocol has demonstrated its efficacy in promoting follicular growth, ovulation, and P/AI, mainly in heifers and anestrous cows. Currently, treatment with recombinant glycoproteins with eCG-like activity (r-eCG) have shown promising results in increasing follicular growth, ovulation, and P/AI in cows submitted to P4/E2 -based protocols. Bovine somatotropin (bST) is a naturally occurring hormone found in cows. Recombinant bovine somatotropin (rbST), produced through genetic engineering techniques, has shown potential in enhancing reproductive outcomes in ruminants. Treatment with rbST has been found to improve P/IA, increase donor embryo production, and enhance P/ET in recipients. The use of recombinant hormones allows to produce non-animal-derived products, offering several advantages in assisted reproductive technologies for ruminants. This advancement opens up new possibilities for improving reproductive efficiency and success rates in the field of animal reproduction.(AU)

Insulin-like growth factor-1 improves in vitro meiotic resumption of dromedary camel (Camelus Dromedarius) oocytes

Despite relatively high maturation rate of in vitro matured oocytes in the dromedary camel, however, blastocyst production is very low after in vitro fertilization (IVF). Herein, the influences of oocyte collection method (follicular aspiration vs slicing; Experiment I), the addition of Insulin-like growth factor I (IGF-I) to the maturation medium (Experiment II) on in vitro maturation (IVM) of oocyte were investigated. Although the nuclear maturation did not differ regardless of collecting method, follicular aspiration led to lower degeneration rates than those in controls (P < 0.05). The percentages of oocytes at MII were greater in the presence of IGF-1 than in its absence (71.9% vs 48.4%, respectively, P<0.05). Additionally, the percentages of degenerated oocytes were higher in the control group compared to oocytes cultured in the presence of IGF-I (23.6% vs 10.4%, respectively, P<0.05). IGF-I treatment improved the quality of MII matured oocytes as evidenced by the decrease of cathepsin B (CTSB) activity, a marker of poor quality oocytes, when compared to control ones (P < 0.05). In conclusion, follicular aspiration decreased the degeneration rate; however, it had no effect on completion of maturation. IGF-I enhanced the IVM of oocyte and decreased degeneration rate.(AU)

Distocia em tigre d'água-de-orelha-vermelha, Trachemys scripta elegans, wied (1839): relato de caso / Dystocia in red-eared slider, Trachemys scripta elegans, wied (1839): case report / Distocia en tortuga de orejas rojas, Trachemys scripta elegans, wied (1839): reporte de caso

Atualmente muitos répteis se tornaram animais de companhia e são mantidos como pet's exóticos. A espécie Trachemys scripta elegans, Wied (1839) é um animal exótico da América do Norte, sua identificação é realizada pelas marcas avermelhadas encontradas lateralmente a sua cabeça. Na rotina clínica as principais enfermidades que acometem os quelônios são as de origem reprodutiva, como a estase folicular e distocia. O objetivo deste trabalho foi relatar um caso recorrente de distocia em um tigre d'água fêmea, para isso, a anamnese, o histórico da paciente, e seus sinais clínicos, em conjunto com os exames complementares de imagem foram essenciais para se obter diagnóstico definitivo. O tratamento foi realizado com a indução medicamentosa utilizando borogluconato de cálcio, seguida da aplicação de ocitocina, esta trouxe resultados positivos para a eliminação dos ovos. Porém devido ao histórico do paciente, optou-se pela intervenção cirúrgica de ovariossalpingectomia, sendo está a maneira permanente de resolução da patologia. O protocolo terapêutico escolhido proporcionou um resultado satisfatório e bem estar ao animal.(AU)

Currently, many reptiles have become companion animals and are kept as exotic pets. The species Trachemys scripta elegans, Wied (1839) is an exotic animal from North America, and its identification is based on the reddish markings found laterally on its head. In routine clinical practice, the main diseases that affect chelonians are those of reproductive origin, such as follicular stasis and dystocia. The aim of this study was to report a recurrent case of dystocia in a female red-eared slider turtle. For this purpose, the patient's anamnesis, history, and clinical signs, along with complementary imaging exams, were essential to obtain a definitive diagnosis. The treatment involved medical induction using calcium borogluconate, followed by the administration of oxytocin, which yielded positive results in egg elimination. However, due to the patient's history, surgical intervention in the form of ovariosalpingectomy was chosen as the permanent solution to the pathology. The chosen therapeutic protocol provided a satisfactory outcome and improved the animal's well-being.(AU)

Actualmente muchos reptiles se han convertido en animales de compañía y se mantienen como mascotas exóticas. La especie Trachemys scripta elegans, Wied (1839) es un animal exótico de América del Norte, su identificación se realiza por las marcas rojizas que se encuentran lateralmente a su cabeza. En la rutina clínica, las principales enfermedades que afectan a los quelonios son las de origen reproductivo, como la estasis folicular y la distocia. El objetivo de este trabajo fue reportar un caso recurrente de distocia en una hembra de tigre de agua, para ello la anamnesis, la historia de la paciente y sus signos clínicos, junto con los exámenes imagenológicos complementarios fueron fundamentales para obtener un diagnóstico definitivo. El tratamiento se realizó con inducción farmacológica con borogluconato de calcio, seguido de la aplicación de oxitocina, que arrojó resultados positivos con la eliminación de huevos. Sin embargo, debido a los antecedentes de la paciente, se optó por la intervención quirúrgica de ovarialpingectomía, que es la forma definitiva de resolución de la patología. El protocolo terapéutico elegido proporcionó un resultado satisfactorio y bienestar al animal.(AU)

Prohibition of hormones in animal reproduction: what to expect and what to do?

As our understanding of ovarian function in cattle has improved, our ability to control it has also increased. The development of Fixed-Time Artificial Insemination (FTAI) protocols at the end of the 20th century has increased exponentially the number of animals inseminated over the last 20 years. The main reasons for this growth were the possibility of obtaining acceptable pregnancy rates without heat detection and, above all, the induction of cyclicity in suckled cows in postpartum anestrus and prepubertal heifers at the beginning of the breeding season. Most FTAI treatments in South America have been based on the use of progesterone (P4) releasing devices and estradiol to synchronize both follicular wave emergence and ovulation, with pregnancy rates ranging from 40 to 60%. These protocols are implemented on a regular basis, allowing producers access to high-quality genetics, and increasing the overall pregnancy rates during the breeding season. In addition, it provided the professionals involved in these programs with a new source of income and the diversification of their practices into activities other than their usual clinical work. Many of these practices are now apparently at risk from restrictions on the use of estradiol by the European Union (EU) and other countries. However, the development of alternative protocols based on GnRH, with P4 devices and eCG and other new products that are not in the market yet will allow us to adapt to the new times that are coming. Logically, the challenge has already been raised and we must learn to use alternative protocols to try to continue increasing the use of this technology in beef and dairy herds. The objective of the present review is to describe the main aspects of banning estradiol in livestock production, the negative impacts on reproductive efficiency, and to present some alternative FTAI protocols for dairy and beef cattle.(AU)

Níveis de progesterona e os parâmetros reprodutivos no período periovulatório de vacas Nelore submetidas ao protocolo de IATF / Progesterone levels and reproductive parameters in the periovulatory period of Nellore cows under FTAI protocol

Background: Fixed Time Artificial Insemination (FTAI) has achieved a significant evolution in the last 18 years, however, despite the progress achieved by modern FTAI programs, the conception rates obtained are still low. Therefore, this study aimed to evaluate the interrelation between progesterone levels in the periovulatory period and reproductive parameters of Nellore cows submitted to an FTAI protocol. Materials, Methods & Results: On a random day, called day 0 (D0), 57 cows received a P4 device associated with the intramuscular (IM) application of 2.0 mg of estradiol benzoate. On D9, the P4 devices were removed and then were administered 500 µg of cloprostenol sodium IM; 0.6 mg of estradiol cypionate IM and 300 IUI of Equine Chorionic Gonadotrophin IM. Blood samples were collected for the determination of serum P4 concentrations on D9 and D11 of the protocol. The evaluations of follicular diameter (DFOL), follicular wall area (AFOL) and the vascularization area of the follicle wall (VFOL) were carried out on D11 using B-mode ultrasonography examination and colour Doppler, and then the artificial inseminations were performed. The evaluation of the corpus luteum diameter (CLD), of the total corpus luteum area (CLA), of the area of corpus luteum vascularization (CLV) and blood sampling for determination of postovulatory P4 levels (Post-P4) were performed on D24. For the analysis of the P4 concentration the chemiluminescence method was used, with a sensitivity of 0.1 ng/mL. According to the P4 concentrations on D11, cows were divided into 2 groups, LOW LEVELS OF P4 and HIGH LEVELS OF P4. The diagnosis of pregnancy was performed using transrectal ultrasonography on D45, at this point the cows were divided into 2 groups, PREGNANT and NON-PREGNANT. The correlation between DFOL and P4 dosage on D11 was moderate, negative and significant and between the AFOL and the serum P4 levels on D9, was moderate, negative and significant. As for the other correlations between follicular and luteal parameters and serum P4 levels, these were low to moderate, negative and not significant. Cows in the LOW LEVELS OF P4 group had significantly larger diameter and follicular areas than the cows in the HIGH LEVELS OF P4 group, the other follicular and luteal parameters showed no statistical difference. Of the total 57 cows that were inseminated, 30 cows became pregnant. Cows in the PREGNANT group had serum P4 levels on D9 equivalent to that obtained by the NON-PREGNANT group. However, at D11 the cows that became pregnant presented significantly lower serum P4 levels than cows that did not become pregnant. Discussion: The results of the interrelation between follicular parameters and P4 levels obtained in the present study, pointed out that the lower the levels of P4, the higher the follicular parameters, corroborating with other authors. Thus, larger preovulatory follicles provided high ovulation rates. Periovulatory serum P4 levels did not significantly affect the morphofunctional parameters of the CL. Such findings may be justified by high periovulatory P4 levels resulting from less efficient luteolysis, exert a negative effect on the results of FTAI protocols, because progesterone inhibits the release of LH pulses. It is concluded that lower periovulatory P4 levels established a favourable condition for follicular development and fertility, however, morphofunctional parameters of the corpus luteum were not affected.

Diet crude protein reduction on follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows

This study evaluated the effect of crude protein (CP) reduction in four diets (156, 139, 132, and 127 g Kg-1 DM) maintaining constant metabolizable protein (188 g/day) on the follicular fluid and cumulus-oocyte complexes of mid-lactating Girolando cows. Twenty-two Girolando cows with average of 21.55 ±3.19 L daily milk yield, 105.30 ±22.62 days in lactation and 3.22 ±0.03 body condition score were selected. To reduce CP in diets and maintain constant metabolizable protein, urea and soybean meal were gradually replaced by lignosulfonate-treated soybean meal (SoyPass®, Cargill), resulting in an increase in rumen-undegradable protein and a reduction in rumen degradable protein. A linear and quadratic reduction was observed in the plasma and follicular fluid urea nitrogen concentration following CP reduction, with the most intense reduction occurring in the 127 g Kg-1 DM group (p<0.001). As CP reduced, there was a tendency for a linear increase in the follicular growth rate (P=0.0696), on the number and proportion of viable oocytes (P<0.09), and also a linear increase for the number (P=0.0397) and proportion (P<0.09) of grade I viable oocytes. Plus, there was a linear effect for the number of cumulus oophorus cells. Cows fed with the lowest amount of CP had cumulus-oocyte complexes with higher numbers of cumulus oophorus cells (P=0.0238). Also, the reduction of diet crude protein was followed by a decrease in the probability of oocytes' DNA degradation. In conclusion, the reduction of CP in the diet of mid-lactating Girolando cows, reduces urea nitrogen concentration in both blood plasma and follicular fluid, and, as a consequence, increases the viability of oocytes and the number of cumulus oophorus cells while reducing oocytes' DNA degradation of follicular included cumulus-oocyte complex. The reduction on dietary CP may improve in vivo oocytes' embryo development impacting fertility of lactating dairy cows.(AU)

Local regulation of antral follicle development and ovulation in monovulatory species

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.(AU)

Regulation of progesterone during follicular development by FSH and LH in sheep

Progesterone (P4) can participate in the development of female mammalian antral follicles through nuclear receptor (PGR). In this experiment, the differences of P4 synthesis and PGR expression in different developmental stages of sheep antral follicles (large > 5mm, medium 2-5mm, small < 2mm) were detected by enzyme-linked immunosorbent assay, immunohistochemistry, qRT-PCR and Western blotting. Secondly, sheep follicular granulosa cells were cultured in vitro. The effects of different concentrations of FSH and LH on P4 synthesis and PGR expression were studied. The results showed that acute steroid regulatory protein (StAR), cholesterol side chain lyase (P450scc) and 3β Hydroxysteroid dehydrogenase (3β-HSD) and PGR were expressed in antral follicles, and with the development of antral follicles in sheep, StAR, P450scc and the expression of 3β-HSD and PGR increased significantly. In vitro experiments showed that FSH and LH alone or together treatment could regulate P4 secretion and PGR expression in sheep follicular granulosa cells to varying degrees, hint P4 and PGR by FSH and LH, and LH was the main factor. Our results supplement the effects of FSH and LH on the regulation of P4 synthesis during follicular development, which provides new data for further study of steroid synthesis and function in follicular development.(AU)

Infuence of heat stress on in vitro oocyte and embryo production in high-yielding Holstein cows / Influência do estresse calórico na produção in vitro de oócitos e embriões de vacas Holandesas de alta produtividade

The objective of this study was to evaluate the influence of thermal shock on oocytes used in the production of in vitro embryos (IVP) of high productivity Holstein cows on the day of follicular aspiration (OPU; 0), 30, 60 and 90 days before the OPU. From the mean temperature on day 0 and on the previous 30, 60 and 90 days, they were classifed into comfort group (TC; up to 15°C) and heat stress (HS; above 15°C) groups.Anegative influence was observed on oocytes and viable embryos (total and grade I). The heat stress in the periods of 30 and 60 days prior to OPU resulted in lower production of viable oocytes (P=0.0028; P=0.0092, respectively). Under stress, on the day of OPU (HS-OPU), cows showed no reduction in the amount of viable oocytes (P=0.5497) and there was no influence of temperature for the group stressed 90 days before OPU (P=0.8287). For total embryos, the difference occurred only in the HS-30 group (P=0.0317), where the groups HS-OPU, HS-60, HS-90 presented, respectively, P=0. 1987, P=0.0596 and P=0.4580. Regarding the production of embryos of grade 1, there was no difference for the groups HS-OPU (P=0.2291) and HS-90 (P=0.2868), but there was a reduction for HS-30 (P=0.0143) and HS-60 (P=0.0253). In summary, heat stress had a negative impact when it occurred 30 or 60 days before follicular aspiration. In addition, 30 days seems to be the period of more susceptibility and that causes the greatest deleterious effects on oocyte viability and IVP.(AU)

Objetivou-se avaliar a infuência do estresse térmico em oócitos utilizados na produção in vitro de embriões (PIV) bovinos da raça Holandesa de alta produtividade no dia da aspiração folicular (OPU; 0), 30, 60 e 90 dias antes da OPU. A partir da temperatura média no dia 0 e aos 30, 60 e 90 dias anteriores, foram classificados nos grupos conforto (CT; até 15°C) e estresse por calor (ET -acima de 15°C). Observou-se infuência negativa em oócitos e embriões viáveis (total e grau I). A submissão ao estresse térmico nos períodos de 30 e 60 dias anteriores à OPU resultou em menor produção de oócitos viáveis (P=0,0028; P=0,0092, respectivamente). Sob estresse, no dia da OPU (ET-OPU), as vacas não apresentaram redução na quantidade de oócitos viáveis (P=0,5497) e não houve infuência da temperatura para o grupo estressado 90 dias antes da OPU (P=0,8287). Para embriões totais, a diferença ocorreu apenas no grupo ET-30 (P=0,0317), onde os grupos ET-OPU, ET-60, ET-90 apresentaram, respectivamente, P=0,1987, P=0,0596 e P=0,4580. Em relação à produção de embriões grau 1, não houve diferença para os grupos ET-OPU (P=0,2291) e ET-90 (P=0,2868), porém houve redução para ET-30 (P=0,0143) e ET- 60 (P=0,0253). Em resumo, o estresse por calor teve impacto negativo quando ocorreu 30 ou 60 dias antes da aspiração folicular. Além disso, 30 dias parece ser o período de maior suscetibilidade e que causa os maiores efeitos deletérios na viabilidade oocitária e na PIV.(AU)

Retrospectiva dos principais biomarcadores envolvidos no desenvolvimento folicular e oocitário em mamíferos / Retrospective of the main biomarkers involved in follicular and oocyte development in mammals

A análise da expressão das diversas proteínas relacionadas a foliculogênese e oogênese in vivo e in vitro, através da proteômica, tem demonstrado possíveis biomarcadores celulares. A descoberta desses biomarcadores pode ajudar na elucidação de mecanismos complexos como a foliculogênese (entendimento sobre o crescimento folicular e maturação oocitária), no tratamento de doenças relacionadas a infertilidade feminina, bem como, no aprimoramento de biotécnicas reprodutivas como a manipulação de oócitos inclusos em folículos pré-antrais. Desta forma, a presente revisão abordou as principais proteínas relatadas nos mais novos trabalhos referentes aos estudos proteômicos visando melhores esclarecimentos referentes ao desenvolvimento folicular e oocitário in vivo e in vitro. A escolha dos artigos foi realizada com base nas descobertas mais atuais e do impacto que determinadas proteínas poderiam trazer para melhorar o entendimento da foliculogênese e oogênese nas diferentes espécies de animais mamíferos.(AU)

The analysis of the expression of several proteins related to folliculogenesis and oogenesis in vivo and in vitro, through proteomics, has demonstrated possible cellular biomarkers. The discovery of these biomarkers may help in the elucidation of complex mechanisms such as folliculogenesis (understanding of follicular growth and oocyte maturation), in the treatment of diseases related to female infertility, as well as in the improvement of reproductive biotechniques such as the manipulation of oocytes enclosed in preantral follicles. Thus, the present review addressed the main proteins reported in the newest works related to proteomic studies aiming at better clarifications regarding follicular and oocyte development in vivo and in vitro. The choice of articles was based on the most current discoveries and the impact that certain proteins could bring to improve the understanding of folliculogenesis and oogenesis in different species of mammalian animals.(AU)

Influence of eCG and reproductive management in the resynchronization of ovulation in dairy goats

Resynchronization protocols have been proposed as a way of shortening females' unproductive time in the flock, with good results in cattle and sheep. In goats, initial studies have shown that a second progestogen device inserted before luteolysis and pregnancy diagnosis does not interfere with the corpus luteum lifespan or functionality. This study aimed to evaluate the follicular growth, ovulation pattern and pregnancy rate after insertion of a second and new progestogen device for resynchronizing, with or without equine Chorionic Gonadotrophin (eCG), submitted to natural mating (NM) or artificial insemination (AI) to propose a viable resynchronization protocol for dairy goats. A total of 38 multiparous Saanen goats underwent a short-term progesterone protocol [six days exposed to medroxyprogesterone acetate (MAP) intravaginal sponges + 200 IU eCG and 0.12 mg of cloprostenol sodium on the 5th day + 0.025 mg of lecirelin 34 hours after sponge withdrawal] and, on day 16th after the ovulation, received a new MAP device which was retained until day 21. At this moment females were split into four groups: GeCG+NM ­ 100 IU eCG with NM; GSal+NM ­ saline solution with NM; GeCG+AI ­ 100 IU eCG with AI; and GSal+AI ­ saline solution with AI. Ultrasound scans were performed every 12 h from sponge withdrawal (day 21) until 108 h after sponge withdrawal (day 25) for follicular dynamics evaluation, at 240 h (day 31) for assessing the presence of active corpus luteum, and on day 60 for pregnancy diagnosis. No differences were found regarding ovulation time, synchronization and follicle size. However, GeCG+NM presented a greater estrus manifestation rate (100%) and pregnancy rate (62.5%) when compared to GSal+AI. In conclusion, resynchronization protocols in dairy goats may present satisfactory results.(AU)

Effects of heat-stress on oocyte number and quality and in vitro embryo production in Holstein heifers

Background: This study aimed to determine the effects of environmental temperature on the number and quality of oocytes and embryo production rates obtained by performing ovum pick up (OPU). Heat stress leads to long-term, short-term, visible, and invisible effects in dairy cows. Its effects on reproduction are evident in all stages, from oocyte development to birth. Disturbance in ovarian follicle development, follicular dominance deficiency, anoestrus, polyspermia, embryonic losses, decreased fetal growth, and abortion are some examples of responses to these effects. The aim of the present study was aimed to determine the effects of ambient temperature on oocyte quality and number and embryo production rates. Materials, Methods & Results: The animal material used in this study comprised 10 Holstein heifers. At the beginning of the study, the heifers were 13-15 months old. OPU was performed at different times of the year, and weather conditions were recorded. Grouping according to ambient temperature was done as < 10°C (group 1), 10-25°C (group 2), and > 25°C (group 3). The veterinary ultrasonography device and a set of compatible intravaginal OPU probe, catheter, and aspiration device were used for OPU application. All antral follicles with diameters of 2-8 mm in the ovaries were aspirated. The aspirated follicle fluids were examined under a stereo microscope, and the cumulus-oocyte complexes (COC) were collected and classified according to their structure. A, B, and C-quality oocytes were included in the in vitro embryo production process. After performing 69 OPUs on random days of the cycle, the number of oocytes per OPU was found to be 8.72, 6.32, and 6.85 in groups 1, 2, and 3, respectively (P < 0.05). The number of viable oocytes per OPU was 6.83, 4.64, and 4.65 in groups 1, 2, and 3, respectively (P < 0.05). The statistical difference between the first group and the other groups was significant for cleavage and blastocyst counts (P < 0.05). Discussion: All the negative effects of heat stress on animals resulted from the increased body temperature. Reproductive performance is adversely affected by high temperatures and humidity during periods of high ambient temperatures. Metabolic heat is released, and the heat load increases due to the metabolism of nutrients in cattle. Internal body temperature is regulated via the dissipation of metabolic heat to the environment. The amount of heat dissipated via conduction and convection depends on the unit body weight, surface area, skin and coat color, difference in temperature gradient of the animal and ambient temperature, and humidity. In the present study, it was determined that the blastocyst development rates of the oocytes obtained in the warm season (>25°C [group 3]) were lower than those of the other groups. It was concluded that this may be because the oocytes developed under chronic heat stress in the animals, and several cycles were required to enhance oocyte quality and developmental potential. Additional studies are needed to investigate the response of oocytes obtained with OPU to heat stress during embryonic developmental stages and to determine the sensitivity and effects of embryonic tissue damage according to developmental stages. Based on the results of the present study, it was concluded that performing OPU and in vitro embryo production (IVEP) when the ambient temperature is close to the thermoneutral limits may increase the blastocyst development rates.

Qualitative and quantitative aspects of atresia during mammalian folliculogenesis / Aspectos qualitativos e quantitativos da atresia durante a foliculogênese em mamíferos

Durante a foliculogênese ovariana, cerca de 99,9% dos folículos morrem pelo processo de atresia folicular. O processo de atresia pode ocorrer pelas vias degenerativa ou apoptótica. Este processo compromete todos os estágios de desenvolvimento folicular, sendo os folículos antrais os mais afetados. Por outro lado, os folículos préantrais são mais resistentes, em função de sua menor taxa metabólica, bem como do número reduzido de células e camadas de células somáticas, células da granulosa e/ou da teca. Embora folículos pré-antrais sejam menos afetados pelo processo de atresia, quando este evento ocorre, pode-se classificá-lo de duas formas, degeneração do tipo I e degeneração do tipo II. Na degeneração do tipo I, o oócito é o compartimento mais comprometido, apresentando núcleo picnótico, embora suas células da granulosa apresentem-se bem organizadas e sem picnose nuclear. Já na degeneração do tipo II, os folículos apresentam oócito retraído e células da granulosa edemaciadas, desorganizadas e sem aderência à membrana basal e ao oócito. É importante destacar que a degeneração do tipo I é mais comum em folículos primordiais e primários, enquanto folículos secundários apresentam mais degeneração do tipo II, ou seja, a medida que os folículos evoluem, a degeneração do tipo II é mais frequente. Considerando todos os aspectos aqui relacionados, essa revisão de literatura abordará aspectos relacionados aos processos de foliculogênese e atresia folicular, bem como as substâncias que induzem a atresia durante a foliculogenese in vitro e in vivo e, ainda, os métodos e parâmetros para análise da atresia em folículos ovarianos. Isto se deve a necessidade de desenvolvimento de protocolos mais eficientes de recuperação dos folículos ovarianos, prevenindo essa grande perda folicular e otimizando as possibilidades de utilização desses materiais biológicos no futuro.

During ovarian foliculogenesis, about 99.9% of follicles die by the follicle atresia process. The atresia process can occur via degeneration or apoptosis. This process compromises all the follicle development stages, with antral follicles being those most affected. On the other hand, the preantral follicles are more residents, since their slow metabolic rate, as well as the reduced number and layers of somatic cells, granulosa and/or thecal cells. Although preantral follicles are less affected by the atresia process, when the event occurs, we can classify it in two ways, type I or type II degenerated follicles. In the type I degeneration the oocyte is the most compromised compartment, showing the picnotic nuclei, although its granulosa cells presented well organized and no picnosis. Already in the type II degeneration, the follicles presented shrunken in the oocyte, and swollen, disorganization and no adhered granulosa cells from the basal membrane and oocyte. It is important to highlight that type I degeneration is the most common in primordial and primary follicles, while in secondary follicles present more type II degeneration, which means that the as follicle evolve, type II degeneration is more frequent. Considering all the aspects related here, this literature review will address aspects related to the folliculogenesis and the process of follicle atresia, as well as substances that induce atresia during in vitro and in vivo folliculogenesis and methods and parameters for analyzing atresia in ovarian follicles. This is due to the need to develop more efficient ovarian follicle recovery protocols, which can prevent this great follicular loss and optimizing the possibilities of use of these biological materials in the future.

Oogênese e Foliculogênese em Bovinos / Oogenesis and Folliculogenesis in Cattle

Os ovários desempenham papéis relevantes para o sistema reprodutivo, no qual a função exócrina (ou gametogênica) realiza a maturação e liberação do oócito para a fecundação e a função endócrina (ou esteroidogênica) afeta a síntese, secreção de hormônios e fatores de crescimento. Além disso, existe uma interação entre os fatores endócrinos, autócrinos e parácrinos, atuando em associação com o processo de desenvolvimento folicular e oocitário durante a vida reprodutiva da fêmea. O córtex, porção mais externa do ovário, representa a região funcional onde se localizam os folículos ovarianos e as estruturas lúteas, em diferentes estágios de desenvolvimento e/ou atresia. A região medular localiza-se mais internamente, na maioria das espécies, e sua principal função é nutrir e sustentar o ovário. Apesar da numerosa população folicular presente nos ovários, estabelecida durante a vida fetal, quase todos os folículos encontrados no pool de reserva ovariana, ou seja, 99,9%, não atingem a ovulação. Esses folículos passam por um processo de morte celular conhecido como atresia folicular, tornando o ovário um órgão de baixa produtividade. A elucidação dos mecanismos que regulam a foliculogênese, incluindo o processo de atresia, é importante para o melhor aproveitamento dos folículos na melhoria da eficiência reprodutiva de animais de produção.

The ovaries play roles relevant to the reproductive system, in which the exocrine (or gametogenic) function carries out the maturation and release of the oocyte for fertilization and the endocrine (or steroidogenic) function effects the synthesis and secretion of hormones and growth factors. In addition, there is an interaction between endocrine, autocrine and paracrine factors, acting in association with the follicular and oocyte development process during the female's reproductive life. The cortex, the outermost portion of the ovary, represents the functional region where the ovarian follicles and luteal structures are located, at different stages of development and/or atresia. The medullary region is located more internally, in most species, and its main function is to nourish and support the ovary. Despite the numerous follicular population present in the ovaries, established during fetal life, almost all follicles found in the ovarian reserve pool, i.e., 99.9%, do not reach ovulation. These follicles undergo a process of cell death known as follicular atresia, making the ovary a low-productivity organ. The elucidation of the mechanisms that regulate folliculogenesis, including the atresia process, is important for the better use of follicles in improving the reproductive efficiency of production animals.

Abordagens genéticas e de bioengenharia para o estudo da foliculogênese / Genetic and bioengineering approaches to the study of folliculogenesis

O desenvolvimento de estudos genéticos e de microdispositivos biológicos tem proporcionado a ampliação do conhecimento sobre os complexos eventos que envolvem a reprodução animal. O desafio ainda é imensurável, mas a criação e surgimentos de novas perspectivas para a pesquisa básica tem-se feito presente. Neste trabalho revisamos de maneira suscinta algumas abordagens recentes, utilizadas pela pesquisa básica, sobretudo com o objetivo de lançar luz sobre o desenvolvimento folicular e oocitário. Dessa forma, essa revisão pretende fornecer uma visão geral do uso das tecnologias ômicas e sistema de microfluídica como auxiliadores na compreensão da foliculogênese. Adicionalmente serão apresentadas particularidades inerentes à fisiologia da gametogênese, que incluem ação de microorganismos e mitocôndrias, além do importante papel da comunicação intercelular através das vesículas extracelulares.

The development of genetic studies and biological microdevices has expanded knowledge about the complex events involving animal reproduction. The challenge is still immeasurable, but the creation and emergence of new perspectives for basic research have been present. This paper briefly reviews some recent approaches used in basic research, mainly to shed light on follicular and oocyte development. Thus, this review intends to provide an overview of the use of omics technologies and microfluidics systems as aids in understanding folliculogenesis. Also, it will present particulars inherent in the physiology of gametogenesis, which include microorganisms and mitochondria, in addition to the important role of intercellular communication through extracellular vesicles.

Comparison of three doses of estradiol benzoate for synchronization of follicular wave emergence in suckled Bos indicus beef cows

We aimed to compare the effect of three estradiol benzoate (EB) doses on follicular wave emergence (FWE) and dominant follicle growth of suckled Nelore cows submitted to TAI (D0). On a random day of estrous cycle (D−10), multiparous (MULT; n=36) and primiparous (PRIM; n=20) suckled Nelore cows received an intravaginal progesterone (P4) device and were assigned in three groups. Cows in the EB-1 (n=20), EB-1.5 (n=15) or EB-2 (n=21) groups received, respectively, an im treatment with 1, 1.5 or 2 mg EB. A subgroup (n=10-13 cows/group) were subject to daily ovarian evaluations from D−10 to D0. On D−2, P4 devices were removed, and all cows received the same treatment: 1 mg estradiol cypionate, 0.53 mg sodium cloprostenol, and 300 IU eCG. Statistical analyses were performed considering only the main effects of treatment group and parity order. The proportion of cows with a synchronized FWE and the moment of the FWE did not differ (p>0.05) among the treatment groups (overall: 80% [28/35] and 4.1 ± 0.4 days); however, the FWE occurred earlier (p=0.007) in MULT (3.8 ± 0.2 days) than PRIM (5.1 ± 0.4) cows. The proportion of animals detected in estrus was greater (86% [31/36] vs. 70% [14/20]; p=0.02) and the dominant follicle was larger on D−2 (9.7 ± 0.3 mm vs. 7.8 ± 0.7 mm; p=0.006) and D0 (11.9 ± 0.4 mm vs. 10 ± 0.5 mm; p=0.008) in MULT than PRIM cows. In conclusion, the three EB doses presented similar efficiency to synchronize the FWE in suckled Nelore cows. Moreover, a delayed FWE and smaller dominant follicle is observed in PRIM cows, contributing to the reduced reproductive performance in this parity category when using similar TAI protocols of MULT cows.

Comparison of three doses of estradiol benzoate for synchronization of follicular wave emergence in suckled Bos indicus beef cows

We aimed to compare the effect of three estradiol benzoate (EB) doses on follicular wave emergence (FWE) and dominant follicle growth of suckled Nelore cows submitted to TAI (D0). On a random day of estrous cycle (D−10), multiparous (MULT; n=36) and primiparous (PRIM; n=20) suckled Nelore cows received an intravaginal progesterone (P4) device and were assigned in three groups. Cows in the EB-1 (n=20), EB-1.5 (n=15) or EB-2 (n=21) groups received, respectively, an im treatment with 1, 1.5 or 2 mg EB. A subgroup (n=10-13 cows/group) were subject to daily ovarian evaluations from D−10 to D0. On D−2, P4 devices were removed, and all cows received the same treatment: 1 mg estradiol cypionate, 0.53 mg sodium cloprostenol, and 300 IU eCG. Statistical analyses were performed considering only the main effects of treatment group and parity order. The proportion of cows with a synchronized FWE and the moment of the FWE did not differ (p>0.05) among the treatment groups (overall: 80% [28/35] and 4.1 ± 0.4 days); however, the FWE occurred earlier (p=0.007) in MULT (3.8 ± 0.2 days) than PRIM (5.1 ± 0.4) cows. The proportion of animals detected in estrus was greater (86% [31/36] vs. 70% [14/20]; p=0.02) and the dominant follicle was larger on D−2 (9.7 ± 0.3 mm vs. 7.8 ± 0.7 mm; p=0.006) and D0 (11.9 ± 0.4 mm vs. 10 ± 0.5 mm; p=0.008) in MULT than PRIM cows. In conclusion, the three EB doses presented similar efficiency to synchronize the FWE in suckled Nelore cows. Moreover, a delayed FWE and smaller dominant follicle is observed in PRIM cows, contributing to the reduced reproductive performance in this parity category when using similar TAI protocols of MULT cows.(AU)

Intravaginal progesterone device (1. 9g) and estradiol benzoate for follicular control in the mare during spring and summer / [Implante intravaginal de progesterona (1, 9g) e benzoato de estradiol no controle folicular de éguas durante a primavera e o verão]

The objective of this study was to evaluate follicular growth and ovulatory rates in mares treated with an intravaginal progesterone device (P4) during the 10-day period, associated with the use of estradiol benzoate (EB). The results were compared during the transition period (ET) in the spring and the breeding season in the summer (ER). The variables were submitted to ANOVA (Tukey's test), considering P<0.05. No ovulation occurred during the permanence of the P4 implant in both experimental periods. The ovulatory rate in the ER was 100% (n = 8) and in the ET 62.5% (n = 5; P = 0.0547). Significant differences were observed (<0.001), in both periods, comparing follicular growth rates during the permanence of P4 device (ER: 1.33 ± 0.89mm/d; ET: 1.00 ± 0.81mm/d) to the period without P4 (ER: 3.63 ± 1.33 mm/d; ET: 3.31 ± 1.66 mm/d). The present study demonstrated applicability and efficiency of a hormonal protocol using P4 intravaginal device and EB for follicular control in mares, both during ET and ER.

O objetivo deste trabalho foi avaliar a taxa de crescimento folicular e a taxa ovulatória em éguas tratadas com dispositivo intravaginal de progesterona (P4) durante o período de 10 dias, associado à utilização de benzoato de estradiol (BE). Os resultados foram comparados durante o período de transição (ET) da primavera com a época de reprodução no verão (ER). As variáveis foram submetidas à ANOVA (teste de Tukey), considerando-se P<0,05. Nenhuma ovulação ocorreu durante a permanência do dispositivo de P4 em ambos os períodos experimentais. A taxa ovulatória na ER foi de 100% (n = 8) e na ET, de 62,5% (n=5; P=0,0547). Diferença significativas (<0,001) foram observadas, em ambos os períodos experimentais, comparando as taxas de crescimento folicular durante a permanência da P4 (ER: 1,33 ± 0,89mm/d; ET: 1,00 ± 0,81mm/d) com o período sem P4 (ER: 3,63 ± 1,33mm/d; ET: 3,31 ± 1,66mm/d). O presente estudo demonstrou aplicabilidade e eficiência do protocolo hormonal utilizando dispositivo intravaginal de P4 e BE para controle folicular de éguas, tanto na ET quanto na ER.

Effects of ergosteroside combined risedronate on fracture healing and BMP-2, BMP-7 and VEGF expression in rats

Purpose To evaluate the effect of ergosterol combined with risedronate on fracture healing. Methods Sixty male Sprague Dawley fracture model rats were assigned into group A (n=20), group B (n=20), and group C (n=20) at random. All rats were fed by gavage until their sacrifice as it follows: group A with ergosteroside and risedronate, group B with risedronate, and group C with saline solution. At weeks 2 and 4, 10 rats of each group were sacrificed. Healing effect and bone tissue changes in the fractures site were assessed by using hematoxylin and eosin stain histology. Enzyme-linked immunosorbent assay was used to detect the expression of serum bone morphogenetic protein-2 (BMP-2), bone morphogenetic protein-7 (BMP-7), and vascular endothelial growth factor (VEGF). Reverse transcriptase polymerase chain reaction was applied to detect the expression of osteoprotegerin (OPG) mRNA, osteocalcin (OCN) mRNA and core-binding factor subunit-?1 (CBF-?1) mRNA. Results In terms of serum BMP-2, BMP-7, and VEGF expression at weeks 2 and 4 after gavage, group A < group B < group C (P<0.05). At week 4 after gavage, serum VEGF expression in the three groups harbored positive relationship with serum BMP-2 and BMP-7 expression (P<0.05). Regarding serum OPG, OCN and CBF-?1 mRNA expression at weeks 2 and 4 after gavage, group A