Pregnancy success in bitches - evaluation of interactions between artificial insemination method, serum progesterone concentration and vaginal cytology parameters

Background: The artificial insemination has become a well-established method in the breeding of bitches, and evaluation of the factors that may potentially affect pregnancy success is essential. For this reason, it is essential to evaluate the factors that may affect fertility of the bitch when artificial insemination is performed. Serum progesterone concentrations and vaginal cytology have been used to determine the time of ovulation and stage of the estrus cycle. This study aimed to evaluate the artificial insemination method, the serum progesterone concentration, the breed size, age, the whelping number, vaginal cytology parameters, and their interactions on pregnancy success in bitches. Materials, Methods & Results: A total of 607 bitches that had undergone reproductive consultation with the Mexican Canine Federation from January to December 2016 were enrolled in the present study and assigned to one of 2 artificial insemination methods (intravaginal and transcervical) using fresh semen. Determination of the estrus cycle phase and the time of Artificial insemination was based on vaginal cytology and serum progesterone concentrations. Bitches inseminated by the transcervical technique had a higher pregnancy rate with respect to females inseminated by the intravaginal technique (P < 0.05). Moreover, females with a serum progesterone concentration of 5-10 ng/mL had a greater probability (> 4 times) of getting pregnant than animals with lower or higher progesterone concentrations (P < 0.05). Bitches inseminated by the intravaginal technique and with serum progesterone concentrations >10 ng/mL had a considerable reduction in pregnancy (P < 0.05) compared with females with < 10 ng/mL serum progesterone or with bitches inseminated by the transcervical technique. Discusion: Serum progesterone concentration, the artificial insemination method, and superficial cells without a nucleus modified the pregnancy rate in bitches. Females inseminated by transcervical semen deposition had a higher pregnancy rate than females inseminated by the intravaginal technique. Using fresh or frozen-thawed semen produced a higher pregnancy rate in bitches inseminated by transcervical semen deposition than females inseminated by the intravaginal technique. Differences in the pregnancy rate between transcervical and intravaginal insemination could be associated with the correct semen disposition, the distance that the sperm must travel to reach the oocyte, as well as the number of sperm that reach the oviduct ampulla. Exist evidences that after ovulation, as progesterone rises, the cervix is closed, which may compromise the passage of the sperm deposited into the vagina. Therefore, it is likely that in females with a serum progesterone concentration > 10 ng/mL, the cervix was closed, compromising the ability of the sperm to access the oviduct. Thus, the use of intravaginal insemination should be done in bitches with a serum progesterone concentrations < 11 ng/mL to reduce the possibility of cervical closure and to increase the odds of pregnancy. It is well documented that the serum progesterone concentration and vaginal cytology parameters have a great influence on pregnancy success, and the results confirm these findings. In the present study, 96% of the bitches inseminated with a serum progesterone concentration of 5-10 ng/mL got pregnant and had higher odds of pregnancy than bitches with lower or higher serum progesterone concentrations.

Gestational follow-up in ewes - placentome size and fecal progesterone metabolite levels

Background: Transabdominal ultrasonography in sheep allows real-time diagnosis and monitoring of pregnancy as well as measurement of the size of placentomes, which are structures that connect the fetal and maternal portions, that increase as pregnancy progresses. Progesterone is involved in the maintenance of gestation, with high levels noted during pregnancy. In this context, it is hypothesized that measurements of placentome and progesterone could help in monitoring pregnancy in sheep. Therefore, the main objectives of this study included evaluating whether placentome size and fecal progesterone levels are associated with gestational age in ewes and whether measurement of the placentome and progesterone could be used as an estimate of gestational age. Materials, Methods & Results: A total of 63 pregnant adult (between 2 and 5 years of age) Santa Inês ewes were monitored in the city of Boa Vista, Roraima. All ewes were kept in the paddock with Panicum maximun cv. tanzania, mineral salt, and water ad libitum, supplemented with 200 g/animal/day of soybean residue. Ewes were subjected to ultrasonography every 21 days from day 42 of pregancy until term to monitor pregnancy. Ultrasonography was performed using a linear probe to measure the placentome, including length × width in centimeters. Also every 21 days, fecal samples were collected for measurement of progesterone level. Feces were collected directly from the rectal ampulla, stored individually in plastic bags, and frozen at -20ºC until processing. In the laboratory, these feces samples were freeze-dried and subjected to hormonal extraction with 80% methanol. Then, the levels of fecal progesterone metabolites were measured by enzyme immunoassay. Data was tabulated and submitted to statistical analyses, including descriptive stastistic, correlation, KruskalWallis test and Tukey's test. The levels of fecal progesterone metabolites increased significantly between the 3rd and 4th months of pregnancy, with a decrease close to delivery. Placentome size increased significantly between the 2nd and 3rd months of pregnancy. Placentome measurements did not correlate with progesterone levels and showed a weak correlation with gestational age. Therefore, we concluded that the measurements of placentome size and a single evaluation of fecal progesterone metabolites were not considered efficient methods for estimating gestational age. Discussion: Measurement of progesterone level in fecal samples is a non-invasive hormone monitoring method that provides values like that of blood levels and is less stressful for the animals at the time of sample collection. The wide physiological variation makes it difficult or even useless to use fecal progesterone, when is measured only once per animal, as an indicator of the presence of pregnancy. Despite this limitation, serial sampling is useful for monitoring of hormones during the pregnancy period and is an important tool for research purposes. Placentome size measurements correlated weakly with gestational age and did not correlate with progesterone level. Thus, the presence of placentomes is indicative of the presence of pregnancy but was considered inefficient for determining gestational age in sheep.

Efeito da progesterona na sincronização de vacas de leite / Effect of progesterone on synchronization of dairy cows

Diversos fatores impactam os índices reprodutivos em vacas leiteiras, como a produção de leite, o manejo nutricional e o programa reprodutivo implementado. Em vacas leiteiras de alta produção, o maior catabolismo de esteróides, como a progesterona, devem ser levados em consideração ao escolher o tratamento hormonal a ser realizado. Assim, estratégias com a suplementação de progesterona previamente e ao início do protocolo foram desenvolvidas e descritas.(AU)

Milk production, nutritional management and the implemented reproductive program impact reproductive rates in dairy cows. The greater catabolism of steroids such as progesterone in high producing dairy cows must be considered when choosing the hormonal treatment to be used. Thus, strategies with progesterone supplementation before and at the beginning of the protocol were developed and described.(AU)

In vivo embryo development in bitches inseminated laparoscopically after ovulation time estimated based on a single progesterone determination

Logistic and economical limitations are often the causes of dog owners not accurately monitoring the estrous cycle and the optimal insemination time. The aim of this study was to evaluate in vivo early embryonic development in bitches, after the analysis of sequential vaginal cytologies associated to single progesterone measurement and single laparoscopic insemination with high quality semen (fresh and with high spermatozoa concentration) or low-quality semen (frozen/thawed and with low spermatozoa concentration) at 48 h post- ovulation time predicted on a single progesterone measurement. Ten bitches were inseminated with 250 x 106 fresh spermatozoa (80% motility), and ten with 80 x 106 frozen/thawed spermatozoa (60% motility) in the cranial part of each uterine horn. Seven days later, ovariohysterectomy was performed and the oviducts and uterine horns and body were flushed to recover embryos and unfertilized oocytes. In 80% of the bitches inseminated with fresh and 50% of bitches inseminated with frozen/thawed semen, embryos at 2 to 8 cells stage were recovered mostly from the, oviducts. This study indicates that pregnancies can be obtained with a single laparoscopic intrauterine insemination after single serum progesterone measurement, although with a low number of embryos. This result should be taken into account in case economic or logistic restrictions that affect the possibility of owners to plan an accurate monitoring of the optimal breeding time using fresh and frozen semen.(AU)

Relationships between follicle and corpus luteum size and vascularization with ovulation, progesterone production, and pregnancy in Nellore beef cattle

ABSTRACT The objective of this study was to evaluate the relationships between preovulatory follicle (POF) and corpus luteum (CL) diameters, and POF and CL vascular perfusion with progesterone production, ovulation, and pregnancy in Nellore cows subjected to timed artificial insemination (TAI). Nellore cows (n = 201) were subjected to ovulation synchronization and later to ultrasound evaluation of POF and CL at the time of insemination (D0) and seven days later (D7), respectively. Females were divided into three categories according to the POF diameter assessed at the time of insemination: small (SF), medium (MF), and large (LF) follicles. The LF group had a greater number and intensity of pixels in the POF ultrasound exam compared with the SF group. The CL flow intensity and progesterone concentration were also higher in the LF group. The SF group showed lower flow intensity and lower ovulation rate compared with the others. When non-pregnant females were compared to pregnant ones, no difference was observed in any of the analyzed variables. The results show for the first time in Nellore cattle the relationship between the size of ovarian structures and blood flow (quantity and intensity) as well as the ability of the CL to produce progesterone. The intensity of the POF pixels proved to be relevant, demonstrating correlations with the size and flow of the CL, which were not found when evaluating only the number of pixels, thus revealing the importance of evaluating complementary characteristics of the flow.

Estratégias farmacológicas para a manipulação do ciclo estral de fêmeas bovinas de raças de corte taurinas / Pharmacological approaches to manipulate bovine estrous cycle in taurine beef breeds

Este texto discute as estratégias farmacológicas para a manipulação do ciclo estral de fêmeas taurinas de corte, com foco na inseminação artificial em tempo fixo (IATF). Os zebuínos, rebanho predominante no Brasil, apresentam características de ciclo estral diferentes das raças taurinas, o que justifica a busca por estratégias hormonais adaptadas para o controle do ciclo estral nas subespécies. O estradiol combinado com a progesterona (P4) e prostaglandina F2 alfa (PGF) é o esquema hormonal mais comumente utilizado para a manipulação do ciclo estral em protocolos de IATF. Porém, o uso de GnRH combinado ou em substituição aos ésteres de estradiol vem sendo considerado. Coletivamente, os dados do nosso grupo reforçam a necessidade de customizar as abordagens para o controle do ciclo estral de acordo com a composição genética das fêmeas bovinas.(AU)

This text discusses pharmacological strategies to manipulate the estrous cycle of taurine and synthetic females, with a focus on timed artificial insemination (TAI). Zebu cattle, the predominant herd in Brazil, have different estrous cycle characteristics than taurine breeds, requiring different synchronization hormonal strategies for each subspecies. Estradiol combined with progesterone (P4) and prostaglandin F2 alpha (PGF) is the most used hormonal scheme for estrous cycle manipulation in TAI protocols. But the use of GnRH instead of estradiol esters is being considered. Collectively, our group's data reinforce the need to customize approaches to estrous cycle control according to the genetic composition of bovine females.(AU)

Clinical and pathological aspects of progressive hyperphosphatasemia associated with vacuolar hepatopathy and hepatocellular carcinoma in a Scottish Terrier bitch / Aspectos clínicos e patológicos da hiperfosfatasemia progressiva associada à hepatopatia vacuolar e carcinoma hepatocelular em uma cadela da raça Scottish Terrier

ABSTRACT: Hyperphosphatasemia refers to an increase in alkaline phosphatase serum activity, and Scottish Terriers (STs) are predisposed to develop this condition of uncertain pathogenesis. This study describes a case of progressive hyperphosphatasemia with vacuolar hepatopathy and hepatocellular carcinoma (HCC) in a ST bitch. This dog had a five-year clinical follow-up with progressive hyperphosphatasemia (up to 5503 U/L) and with ultrasound findings and histologic diagnosis of vacuolar hepatopathy, in addition to posterior onset of HCC. A steroidogenic adrenal panel revealed an increase of adrenocortical hormones, especially progesterone and androstenedione, consistent with a subdiagnosed hypercortisolism. Euthanasia was elected and at necropsy, multinodular, yellow to red masses were observed in the liver, which were histologically and immunohistochemically defined as HCC. The association of the clinical, imaging, biochemical, adrenal panel and pathologic findings allowed to characterize and confirm a progressive disorder in this ST bitch associated with elevated adrenocortical hormones.

RESUMO: Hiperfosfatasemia é o aumento sérico de fosfatase alcalina, sendo que Scorrish Terriers estão predispostos a desenvolverem essa condição de patogênese desconhecida. Este trabalho descreve um caso de hiperfosfatasemia progressiva com hepatopatia vacuolar e carcinoma hepatocelular em um canino da raça Scottish Terrier. Uma cadela Scottish Terrier foi acompanhada clinicamente por cinco anos devido à hiperfosfatasemia persistente (até 5503 U/L), com achados ultrassonográficos e histológicos compatíveis com hepatopatia vacuolar, além de posterior desenvolvimento de carcinoma hepatocelular. O painel esteroidogênico realizado indicou aumento dos hormônios adrenocorticais, principalmente progesterona e androstenediona, consistente com diagnóstico de hipercortisolismo subdiagnosticado "atípico". Devido ao prognóstico desfavorável, a eutanásia foi realizada e na necropsia, massas amarelas a vermelhas e multinodulares foram observadas no fígado, com diagnóstico de carcinoma hepatocelular pela análise histológica e imuno-histoquímica. A associação dos achados clínicos, de imagem, bioquímicos, do painel androgênico e patológicos permitiram caracterizar e confirmar um distúrbio progressivo no canino da raça Scottish Terrier associado ao aumento dos hormônios adrenocorticais.

Reproductive and economic evaluation of sexual stimulation during the anestrous period in a commercial farm with neonatal lamb losses / Avaliação reprodutiva e econômica da estimulação sexual durante o período antrópico em uma fazenda comercial com perdas neonatais de cordeiros

This study was performed during the anestrous, involving 140 Akkaraman Kangal ewes whose lambs had died in the neonatal stage due to pneumonia and enteritis. Intravaginal sponge containing progesterone was placed to the animals (Group 1, n = 70) on day 0 and removed after 7 days, following which 263 µg PGF2α and 500 IU eCG were administered to the sheep. Ram introduction was performed for 7 days (days 8-14), starting from the day after the removal of the intravaginal sponge (day 8). The animals in Group 2 (n = 70) were not exposed to any treatment. Ram introduction was performed simultaneously in both the groups. To determine the reproductive response, reproductive parameters such as estrous, pregnancy, multiple pregnancy, and embryonic mortality rates, number of births, number of offspring, and fertility, as well as their economic implications, were compared between groups. Each reproductive parameter exhibited a statistical difference between groups. An economically positive trend was observed in the study group compared with the control group. It was concluded that in case of lamb losses in commercial farms that derive profit from lambing, pregnancy of ewes can be achieved via sexual stimulation without waiting for the next breeding season.

Este estudo foi realizado durante o anestrous, envolvendo 140 ovelhas Akkaraman Kangal cujos cordeiros haviam morrido no estágio neonatal devido a pneumonia e enterite. A esponja intragaginal contendo progesterona foi colocada aos animais (Grupo 1, n = 70) no dia 0 e removida após 7 dias, após os quais 263 µg PGF2α e 500 UI eCG foram administrados às ovelhas. A introdução do carneiro foi realizada por 7 dias (dias 8-14), a partir do dia seguinte à remoção da esponja intravaginal (dia 8). Os animais do Grupo 2 (n = 70) não foram expostos a nenhum tratamento. A introdução do carneiro foi realizada simultaneamente em ambos os grupos. Para determinar a resposta reprodutiva, foram comparados entre os grupos parâmetros reprodutivos, tais como estrogênio, gravidez, gravidez múltipla e taxas de mortalidade embrionária, número de nascimentos, número de descendentes e fertilidade, bem como suas implicações econômicas. Cada parâmetro reprodutivo exibia uma diferença estatística entre os grupos. Uma tendência economicamente positiva foi observada no grupo de estudo em comparação com o grupo de controle. Concluiu-se que no caso de perdas de cordeiros em fazendas comerciais que obtêm lucros com a parição, a gravidez de ovelhas pode ser obtida através de estimulação sexual sem esperar pela próxima estação de reprodução.

Atualizações no controle do ciclo estral em ovinos / Updates on the control of the estrous cycle in sheep

Nos últimos anos, observou-se um aumento no tamanho do rebanho ovino no país, indicando uma retomada na ovinocultura. Para melhorar a eficiência reprodutiva desses animais, a utilização de biotécnicas tem se mostrado uma ferramenta importante. Dentre essas biotécnicas, destacam-se os protocolos de sincronização do ciclo estral, os quais frequentemente utilizam progesterona associada a gonadotrofinas ou análogos da prostaglandina em duas aplicações. Este artigo tem como objetivo revisar os protocolos de sincronização utilizados em ovinos e apresentar atualizações relevantes sobre os mesmos.(AU)

In recent years, there has been an increase in the size of the sheep herd in the country, indicating a recovery in sheep farming. To improve the reproductive efficiency of these animals, the use of biotechniques has been shown to be an important tool. Among these biotechniques, estrous cycle synchronization protocols stand out, which frequently use progesterone associated with gonadotropins or prostaglandin analogues in two applications. This article aims to review the synchronization protocols used in sheep and present relevant updates about them.(AU)

Clinical and pathological aspects of progressive hyperphosphatasemia associated with vacuolar hepatopathy and hepatocellular carcinoma in a Scottish Terrier bitch / Aspectos clínicos e patológicos da hiperfosfatasemia progressiva associada à hepatopatia vacuolar e carcinoma hepatocelular em uma cadela da raça Scottish Terrier

Hyperphosphatasemia refers to an increase in alkaline phosphatase serum activity, and Scottish Terriers (STs) are predisposed to develop this condition of uncertain pathogenesis. This study describes a case of progressive hyperphosphatasemia with vacuolar hepatopathy and hepatocellular carcinoma (HCC) in a ST bitch. This dog had a five-year clinical follow-up with progressive hyperphosphatasemia (up to 5503 U/L) and with ultrasound findings and histologic diagnosis of vacuolar hepatopathy, in addition to posterior onset of HCC. A steroidogenic adrenal panel revealed an increase of adrenocortical hormones, especially progesterone and androstenedione, consistent with a subdiagnosed hypercortisolism. Euthanasia was elected and at necropsy, multinodular, yellow to red masses were observed in the liver, which were histologically and immunohistochemically defined as HCC. The association of the clinical, imaging, biochemical, adrenal panel and pathologic findings allowed to characterize and confirm a progressive disorder in this ST bitch associated with elevated adrenocortical hormones.

Hiperfosfatasemia é o aumento sérico de fosfatase alcalina, sendo que Scorrish Terriers estão predispostos a desenvolverem essa condição de patogênese desconhecida. Este trabalho descreve um caso de hiperfosfatasemia progressiva com hepatopatia vacuolar e carcinoma hepatocelular em um canino da raça Scottish Terrier. Uma cadela Scottish Terrier foi acompanhada clinicamente por cinco anos devido à hiperfosfatasemia persistente (até 5503 U/L), com achados ultrassonográficos e histológicos compatíveis com hepatopatia vacuolar, além de posterior desenvolvimento de carcinoma hepatocelular. O painel esteroidogênico realizado indicou aumento dos hormônios adrenocorticais, principalmente progesterona e androstenediona, consistente com diagnóstico de hipercortisolismo subdiagnosticado "atípico". Devido ao prognóstico desfavorável, a eutanásia foi realizada e na necropsia, massas amarelas a vermelhas e multinodulares foram observadas no fígado, com diagnóstico de carcinoma hepatocelular pela análise histológica e imuno-histoquímica. A associação dos achados clínicos, de imagem, bioquímicos, do painel androgênico e patológicos permitiram caracterizar e confirmar um distúrbio progressivo no canino da raça Scottish Terrier associado ao aumento dos hormônios adrenocorticais.

Fatores que impactam na eficiência reprodutiva de vacas de corte / Factors that impact the reproductive efficiency of beef cows

A maior produtividade na pecuária está correlacionada com a eficiência reprodutiva do rebanho. As biotécnicas da reprodução, principalmente os protocolos de sincronização da ovulação, são de grande importância para otimizar a produção e acelerar o ganho genético em rebanhos de corte e de leite. No entanto, alguns fatores como a categoria animal, o manejo reprodutivo implementado na propriedade e o reprodutor utilizado podem afetar os resultados de fertilidade nos protocolos de IATF. Dessa maneira, algumas estratégias foram desenvolvidas para contornar alguns desafios inerentes a vacas de corte em anestro pós-parto e aumentar a eficiência reprodutiva dessas fêmeas. Dentre as estratégias, destacam-se a utilização de progesterona e a suplementação energética/proteica previamente ao início do protocolo de IATF.(AU)

The reproductive efficiency of the herd is correlated with higher productivity in livestock. Reproduction biotechniques as ovulation synchronization protocols are important to optimize production and accelerate genetic profit in beef and dairy herds. However, some factors such as the animal category, the reproductive management implemented on the farm and the sire used can affect the fertility results in the TAI protocols. In this way, some strategies were developed to overcome some challenges of postpartum anestrus beef cows and to increase the reproductive efficiency of these cows. The use of progesterone and energy/protein supplementation before starting the TAI protocol stand out are the strategies.(AU)

Pyometra in queens - changes in haemato-biochemical parameters

Background: Pyometra is a bacterial and hormone-induced reproductive disease that occurs in the post-estrus luteal phase in intact queens. Pyometra is more common in the diestrus period due to the high progesterone concentration (in queens that mated, spontaneously ovulated, or were induced to ovulate). However, it can also be seen due to the use of exogenous hormones such as progesterone for the suppression of estrus. More research is needed in cases of pyometra in queens, as well as in bitches. Because, considering that the pathogenesis and characteristics of feline pyometra is similar to bitches, studies on pyometra-affected bitches are taken as reference in studies and applications on queens. From this point of view, the aims of this study were to reveal the changes in complete blood count, blood gas, and serum biochemistry parameters in feline pyometra cases and to determine the correlation between the mentioned parameters. Materials, Methods & Results: In the study, a total of 25 female cats of different breeds were used, between the ages of 6 months and 7 years, 15 were diagnosed with pyometra, and 10 healthy. Anamnesis, clinical findings, and ultrasonographic examinations were used in the diagnosis of pyometra. Abdominal ultrasonography was performed on queens brought to the clinic with complaints such as anorexia, polydipsia, polyuria, abdominal tension, and fever. The control group (n =10) consisted of queens that were introduced to the clinic and were reproductively healthy. Before any treatment in queens with pyometra and the control group, 1 mL blood samples were taken from v. cephalica to evaluate complete blood count, blood gases and serum biochemistry parameters. In complete WBC, Lym, Mon, Gra, RBC, Hb, HCT, MCV, MCH and PLT parameters and, blood gas parameters such as pH, pCO2 , pO2 , sO2 , Na, K, Cl, lactate, glucose, HCO3 , and BE were also evaluated in taken blood samples. Biochemical parameters BUN, creatinine, ALT, AST, ALP, amylase, T.BIL, D.BIL, P, CHOL, TG, LDH, TP, CPK, ALP, Ca, GGT were measured in serum samples. After examination and laboratory analysis, ovariohysterectomy was performed on queens as a treatment. Granulocyte, WBC, HCT and MCH levels of the pyometra group were higher (P < 0.05) and Lym levels were lower (P < 0.05) compared to the control group. According to these results, pH, HCO3 , and BE were lower (P < 0.05) in queens with pyometra than those in the control group, while Na and lactate parameters were higher (P < 0.05). According to the results of biochemical analysis, it was determined that BUN, creatinine levels, GGT, and LDH enzyme activities were found to be higher in the pyometra group compared to the control group, while the Ca level was found to be low (P < 0.05). A positive correlation was observed between BUN and creatinine and LDH, WBC, granulocyte, HCT, and lactate, and a negative correlation between lymphocytes, pH, and BE in the correlation analysis performed on queens with pyometra and control group. However, a positive correlation was observed between creatinine and LDH and HCT, and a negative correlation between lymphocyte, pH and BE. Discussion: There is not enough information about pyometra in queens. As a result, it was determined that there were significant changes in complete blood count, blood gases and serum biochemical parameters in queens with pyometra in this study. These changes were generally thought to be related to dehydration and sepsis or endotoxemia. In addition, it was evaluated that prerenal azotemia occurring in pyometra affected queens may cause renal dysfunction. For this reason, it is thought that the results obtained in the presented study may contribute to the diagnosis, treatment, and prognosis of pyometra cases in queens.

Reproductive behavior of Pantaneiros sheep, Mato Grosso do Sul, Brazil

The objective was to evaluate fertility, cyclicality behavior, and sexual performance of Pantaneiro sheep throughout the year's photoperiods in Mato Grosso do Sul - Brazil. Two experiments were conducted. In experiment one, the ewes used aged between 2 and 4 years, distributed in three homogeneous groups of 54 animals and subjected to reproductive evaluation in three different predefined luminosity periods, consisting of autumn (11h 45 min.), spring (12h 30 min.), and summer (12h 55 min.). The characteristics studied were body weight (BW), body condition score (BCC), estrus distribution (EST), percentage of pregnant females per cycle (%P), plasma progesterone (PP), and fertility (F) in the three predefined seasons. The mating system used was controlled natural nocturnal and lasted for 45 days. Experiment two, evaluated the ram sexual activity. The males (10) were individually exposed to four ovariectomized ewes. The ewes cycled during evaluated seasons, and the highest fertility was observed in animals with BCC >2.75. The PP concentration differed among periods, and higher conception rates (F) were observed in autumn and spring. The rams (75%) had a medium of sexual performance when they performed more than two ejaculations in 20 min throughout the seasons. Pantaneiro sheep of both genders had a similar fertility index, with early and constant activity throughout the year, displaying no photoperiodism or seasonality.

Importance of preovulatory estradiol on uterine receptivity and luteal function

Animals that exhibited estrus had greater pregnancy success compared to animals that did not exhibit estrus before fixed-time AI (FTAI). Estradiol is synthesized in bovine ovarian follicles under gonadotropin regulation and can directly and indirectly regulate the uterine receptivity and luteal function. Estradiol concentrations at FTAI impacted oviductal gene expression and has been reported to play an important role in establishing the timing of uterine receptivity. These changes have been reported to impact uterine pH and sperm transport to the site of fertilization. After fertilization, preovulatory estradiol has been reported to improve embryo survival likely by mediating changes in uterine blood flow, endometrial thickness and changes in histotroph. Cows with greater estradiol concentrations at the time of GnRH-induced ovulation also had a larger dominant follicle size and greater circulating progesterone concentrations on day 7. Therefore, it is impossible to accurately determine the individual benefit of greater estradiol concentrations prior to ovulation and greater progesterone concentrations following ovulation to pregnancy establishment, as these two measurements are confounded. Research has indicated an importance in the occurrence and timing of increasing preovulatory concentrations of estradiol, but increasing estradiol concentrations by supplementation may not be sufficient to increase fertility. Increased production of estradiol by the preovulatory follicle may be required to enhance fertility through the regulation of sperm transport, fertilization, oviductal secretions, the uterine environment, and embryo survival.(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)

Early maturation of corpus luteum in rabbits - effect of sildenafil citrate on luteolytic capacity in the early luteal period

Background: Prostaglandin F2 alpha (PGF2 α) binds to the specific receptor (PTGFR) on the corpus luteum (CL) in mammals, inducing regression of the CL structure (luteolysis) and initiating a new cycle. While PGF2 α is effective only on mature CL, the immature CL structure (early luteal phase) resists PGF2 α. In this study, sildenafil citrate, which is used to increase blood flow in the genital organs for treating specific pregnancy issues in women, was administered during the early luteal phase in a rabbit model to test the hypothesis of enhancing blood flow to the CL, thereby promoting earlier maturation and enabling a response to PGF2 α. Materials, Methods & Results: The study was conducted in 2 sub-studies: clinical and molecular. A large number of rabbits were initially included in the sub-studies to ensure a sufficient number of pseudo-pregnant rabbits. Ovulation in rabbits was induced with buserelin acetate and was considered as day 0 of the study. The sub-studies were continued with rabbits whose pseudo-pregnancies were confirmed according to progesterone (P4 ) results. As a result, the studies were continued with a total of 41 pseudo-pregnant New Zealand female rabbits, 21 of which were included in the clinical sub-study and 20 in the molecular sub-study. In both sub-studies, on day 3 of the luteal period, rabbits in the treatment group received 5 mg/kg sildenafil citrate and all rabbits received a single dose of exogenous PGF2 α on day 4 to induce luteolysis. In the clinical sub-study, echotexture and intraovarian blood flow changes in the ovaries were determined by ultrasonography (USG) examination. In the molecular sub-study, the expression changes of Hypoxia Inducible Factor 1 Alpha (HIF1A) and Vascular Endothelial Growth Factor (VEGF) related to angiogenesis, Steroidogenic Acute Regulatory Protein (StAR) related to P4 metabolism, Prostaglandin-Endoperoxide Synthase 2 (PTGS2) related to prostaglandin (PG) mechanism and 15-Hydroxyprostaglandin Dehydrogenase (HPGD) genes at mRNA level were determined using Real Time Polymerase Chain Reaction (RT-PCR) in CL tissues obtained with ovariohysterectomy (OVH) at 1 and 12 h after PGF2 α injection. In addition, blood samples were collected for determine P4 levels from all rabbits. In the clinical sub-study; there was no difference between the groups in mean gray values (MGV), whereas there was a significant decrease in both pulsatile index (PI) and resistance index (RI) values at 40 min after PGF2 α injection (P < 0.05). In the molecular sub-study, it was determined that sildenafil citrate had no significant effect (P > 0.05) on the expression levels 1 and 12 h after PGF2 α injection. According to the results of the molecular sub-study, no significant effect of sildenafil citrate on the mRNA expression levels in the investigated genes was detected (P > 0.05). However, within each group, differences were found according to OVH time after PGF2 α injection. It was observed that PTGS2 and HPGD mRNA expressions decreased at the 12th h compared to the 1st h, while HIF1A expression increased (P < 0.05). Discussion: According to the results obtained from clinical and molecular sub-studies, it was determined that a single dose of sildenafil citrate (5 mg/kg) applied on the 3rd day of the luteal period did not contribute to the maturation process of the CL, did not increase blood flow, and was insufficient to break the resistance of the CL against PGF2 α applied on the 4th day of the luteal period. However, a significant decrease in the PI value at the 40th min after PGF2 α injection suggests that sildenafil citrate has a supportive effect, and that this decrease is also seen in the RI value, suggesting that its effect is insufficient against the vasoconstrictive effect of PGF2 α.

Evaluation of the use of dry blood spots for progesterone determination in ewes / Avaliação do uso de manchas secas de sangue para dosagem de progesterona em ovinos

The use of dried blood spots on filter paper has been shown to be a practical alternative in several studies with humans and animals, enabling a simple means of storing and transporting viable blood samples for various laboratory analyses. However, its applicability in the measurement of progesterone in animals is scarce. Thus, the objective of this study was to evaluate the feasibility of using dried blood spots for the measurement of progesterone in sheep. In total, 38 blood samples from 6 sheep were dripped onto filter paper, and the remainder of each sample was separated into serum. The progesterone levels in the serum samples and in the dry drops were measured by enzyme immunoassay and subsequently correlated. The levels of progesterone in the serum and dry spots showed a high correlation between the matrices (R2 = 0.9694). In conclusion, this study demonstrated the feasibility of using samples of dried sheep blood spots for the measurement of progesterone, and the storage and transport technique can be applied in the field.

O uso de manchas secas de sangue em papel filtro tem se demonstrado uma alternativa prática em vários estudos com humanos e animais, possibilitando um meio simples de armazenamento e transporte de amostras de sangue viáveis para várias análises laboratoriais. Entretanto, sua aplicabilidade na dosagem de progesterona em animais é escassa. Deste modo, o objetivo deste estudo foi avaliar a viabilidade do uso de manchas secas de sangue para a dosagem de progesterona em ovinos. Assim, 38 amostras de sangue de 6 ovelhas foram gotejadas em papel filtro e o restante de cada amostra foi separado o soro. Os níveis de progesterona nas amostras de soro e nas gotas secas foram mensurados por enzimaimunoensaio e posteriormente correlacionados. Os níveis de progesterona no soro e nas manchas secas apresentaram uma alta correlação entre as matrizes (R2=0,9694). Em conclusão, este estudo demonstrou a viabilidade do uso de amostras de manchas secas de sangue de ovino para a dosagem de progesterona, podendo a técnica de armazenamento e transporte ser aplicada a campo.

Efeitos da progesterona injetável em vacas de corte com diferentes escores de cio antes do início do protocolo de IATF / Effects of injectable progesterone in beef cows with different etrus scores before the start of the FTAI protocol / Efectos de la progesterona inyectable en vacas de carne con diferentes puntuaciones de estro antes del inicio del protocolo IATF

A IATF é uma biotecnologia da reprodução que permite o melhoramento genético dos rebanhos de corte, pois possibilita a sincronização da ovulação e realização da inseminação artificial sem necessidade de detecção do cio. O objetivo deste estudo, foi avaliar a eficiência da pré-sincronização com a utilização prévia de progesterona (P4) injetável, sua influência e efeitos na taxa de prenhez, por meio da realização de um protocolo de IATF em vacas de corte multíparas, em quatro fazendaslocalizadas no Mato Grosso do Sul-SP. Para o estudo foram utilizadas 396 vacas nelores, os animais de cada fazenda foram alocados em grupos distintos experimental e controle. Foram avaliadas 196 vacas pertencentes ao grupo controle, e 200 vacas tratadas com progesterona no total, sendo as fazendas descritas neste estudo como fazenda A, B, C e D. Dez dias antes do início do protocolo (D-10), foi aplicado progesterona injetável, via intramuscular em animais do mesmo lote, no D0 foi aplicado benzoato de estradiol e colocado o dispositivo intravaginal com liberação de progesterona, além da classificação dos animais que apresentavam ou não o corpo lúteo. No D8 o dispositivo foi retirado e aplicado uma dose de gonadotrofina coriônica equina, e cipionato de estradiol e de prostaglandina. A inseminação artificial em tempo fixo foi realizada no D10, com a utilização de sêmen de touros provados. O diagnóstico de gestação foi feito no D40, comparando os resultados das taxas de prenhez das vacas dos diferentes grupos, que foram tratadas ou não com progesterona de forma prévia ao protocolo. Sobre as taxas de prenhez dos animais tratados com progesterona, foram encontrados os resultados da fazenda A= 6,60%, fazenda B=10,80%, fazenda C= 3,90% e fazenda D= 3,70%, assim ficando evidente que essa biotecnologia da reprodução é lucrativa e contribui positivamente para a cadeia de produção de bovinos de corte. Baseado nos resultados, pode-se concluir que o uso da P4 (injetável) obteve êxito no índice de prenhez.(AU)

The IATF is a reproduction biotechnology that allows the genetic improvement of beef herds,as it allows the synchronization of ovulation and artificial insemination without the need for heat detection. The objective of this study was to evaluate the efficiency of pre-synchronization with the previous use of injectable progesterone (P4), its influence and effects on the pregnancy rate, through the performance of an IATF protocol in multiparous beef cows, in four farms located in Mato Grosso do Sul-SP. For the study, 396 Nelore cows were used, the animals from each farm were allocated into different experimental and control groups. A total of 196 cows belonging to the control group were evaluated, and 200 cows treated with progesterone in total, being the farms described in this study as farms A, B, C and D. Ten days before the beginning of the protocol (D-10), injectable progesterone was administered , intramuscularly in animals of the same batch, on D0 estradiol benzoate was applied and the intravaginal device was placed with progesterone release, in addition to the classification of animals that had or did not have the corpus luteum. On D8, the device was removed and a dose of equine chorionic gonadotropin, estradiol and prostaglandin cypionate was applied.Fixed-time artificial insemination was performed on D10, using semen from proven bulls. The pregnancy diagnosis was made on D40, comparing the results of the pregnancy rates of the cows of the different groups, which were treated or not with progesterone prior to the protocol. From the pregnancy rates of animals treated with progesterone, the results were found for farm A= 6.60%, farm B= 10.80%, farm C= 3.90% and farm D= 3.70%, thus becoming evident that this reproductive biotechnology is profitable and contributes positively to the beef cattle production chain. Based on the results, it can be concluded that the use of P4 (injectable) was successful in the pregnancy rate.(AU)

La IATF es una biotecnología de la reproducción que permite la mejora genética de rebaños de carne, ya que permite sincronizar la ovulación y la inseminación artificial sin necesidad de detección de celo. El objetivo de este estudio fue evaluar la eficiencia de la presincronización con el uso previo de progesterona inyectable (P4), su influencia y efectos en la tasa de preñez, mediante la realización de un protocoloIATF en vacas de carne multíparas, en cuatro fincas ubicadas en Mato Grosso do Sul-SP. Para el estudio se utilizaron 396 vacas Nelore, los animales de cada finca se distribuyeron en diferentes grupos experimentales y de control. Se evaluaron un total de 196 vacas pertenecientes al grupo control, y 200 vacas tratadas con progesterona en total, siendo las fincas descritas en este estudio como fincas A, B, C y D. Diez días antes del inicio del protocolo (D-10), se administró progesterona inyectable, por vía intramuscular en animales del mismo lote, sobre D0 se aplicó benzoato de estradiol y se colocó el dispositivo intravaginal con liberación de progesterona, además de la clasificación de animales que tenían o no cuerpo lúteo. El D8 se retiró el dispositivo y se aplicó una dosis de gonadotropina coriónica equina, estradiol y cipionato de prostaglandina. Se realizó inseminación artificial a tiempo fijo en D10, utilizando semen de toros probados. El diagnóstico de preñez se realizó el D40, comparando los resultados de las tasas de preñez de las vacas de los diferentes grupos, que fueron tratadas o no con progesterona previo al protocolo. A partir de las tasas de preñez de los animales tratados con progesterona se encontraron los resultados para la finca A= 6.60%, finca B= 10.80%, finca C= 3.90% y finca D= 3.70%, evidenciándose así que esta biotecnología reproductiva es rentable y contribuye positivamente a la cadena productiva del ganado vacuno de carne. Con base en los resultados, se puede concluir que el uso de P4 (inyectable) fue exitoso en la tasa de embarazo.(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)

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.