Effect of pre-treatment with deslorelin on the ovarian response of ewes superovulated with FSH.

This study evaluated the use of the GnRH agonist hormone, deslorelin, to control the follicular population before initiating multiple ovulation and embryo transfer (MOET) treatment. Twenty-four cross-bred Santa Inês ewes, aged between 2 and 4 years, were randomly assigned to either a control group (n = 11) or a treated group (n = 13). All ewes received an intravaginal device containing 60 mg of medroxyprogesterone acetate on day 0, and a new device on day 7, which remained in place until day 14. Additionally, the ewes were administered 125 µg of cloprostenol on day 7. The superovulatory treatment involved administering 200 mg of pFSH, divided into eight decreasing doses at 12-h intervals starting on day 12. On day 14, 300 IU of eCG was administered. In the deslorelin group, three doses of 100 µg of deslorelin were administered starting on day 3 after the insertion of the vaginal device, with subsequent doses given at 72-h and 144-h intervals. Natural mating was performed 36 h after the removal of the progesterone implant using males with proven fertility. Embryo collection took place on the 6th day after mating, and the recovered structures were quantified and evaluated for quality and developmental stage. Transrectal ultrasonography was conducted on days 12, 16 and 21 to evaluate the ovaries, specifically to assess the ovarian follicular population and the presence of the corpus luteum. Ewes in the control group had higher embryo recovery rates (p < .01) compared to the treated group (5.2 ± 0.8 vs. 1.1 ± 0.8), with differences observed primarily in the number of morulae. The number of corpus luteum observed during the laparotomy on day 21 was significantly higher (p < .01) in the control group (10.44 vs. 4.5 corpus luteum per ewe). Yet, the treated group had a significantly higher number of follicles (p < .05) on the first day of pFSH application (5.5 vs. 3.0 follicles per ewe). In conclusion, although the inclusion of deslorelin in the superovulation protocol resulted in increased synchronization of oestrus and follicle number, it did not lead to an increase in the number of corpus luteum or harvested embryos.

The use of sodium caseinate in the freezing of sheep semen.

The aim of this study was to assess the addition of 2% sodium caseinate in a commercial egg yolk-based medium in frozen ovine semen. Eight Dorper males were used for the study. The ejaculate was divided into two portions and frozen without (G1) or with the addition of 2% sodium caseinate (G2). Kinetic parameters were evaluated using CASA (computer-assisted sperm analysis), and membrane and acrosome integrity as well as oxidative stress were assessed using flow cytometry. After thawing, a thermoresistance test was conducted at time points T0 and T90. For the fertility test, 100 ewes were inseminated with semen from two rams selected based on in vitro parameters, one with good post-thaw quality (+70% total motility) and the other with low post-thaw quality (-55% total motility). For the fertility test, the females were divided into 4 groups for insemination: low-quality ram without caseinate (GBS = 25) and with caseinate (GBC = 25), and high-quality ram without caseinate (GAS = 25) and with caseinate (GAC = 25). Regarding the results of sperm kinetics, there was a statistically significant difference in the parameters of average path velocity (VAP) and curvilinear velocity (VCL) between the group frozen with BotuBov and the group with added caseinate. At time point T90, straight-line velocity maintained a trend (p < .06), with BotuBov® (BB group) being superior to caseinate this time, and in the linearity parameter, caseinate was superior to BotuBov®. Flow cytometry analysis showed no difference between any of the evaluated tests. In the fertility test, there was no statistically significant difference in the pregnancy rate between the BotuBOV® group (23%, 11/48) and the sodium caseinate group (BC group) (33%, 17/52), and no differences were observed in the male versus diluent interaction (p = .70). In conclusion, sodium caseinate supplementation did not influence sperm kinetic parameters and the fertility of sheep.

Uterine secretome: What do the proteins say about maternal-fetal communication in buffaloes?

The aim was to compare the UF proteomics of pregnant and non-pregnant buffalo during early pregnancy. Forty-four females were submitted to hormonal estrus synchronization and randomly divided into two groups: pregnant (n = 30) and non-pregnant (n = 14). The pregnant group was artificially inseminated and divided into a further two groups: P12 (n = 15) and P18 (n = 15). Conceptus and uterine fluid samples were collected during slaughter at, respectively, 12 and 18 days after insemination. Of all the inseminated females, only eight animals in each group were pregnant, which reduced the sample of the groups to P12 (n = 8) and P18 (n = 8). The non-pregnant group was also re-divided into two groups at the end of synchronization: NP12 (n = 7) and NP18 (n = 7). The UF samples were processed for proteomic analysis. The results were submitted to multivariate and univariate analysis. A total of 1068 proteins were found in the uterine fluid in both groups. Our results describe proteins involved in the conceptus elongation and maternal recognition of pregnancy, and their action was associated with cell growth, endometrial remodeling, and modulation of immune and antioxidant protection, mechanisms necessary for embryonic maintenance in the uterine environment. SIGNIFICANCE: Uterine fluid is a substance synthesized and secreted by the endometrium that plays essential roles during pregnancy in ruminants, contributing significantly to embryonic development. Understanding the functions that the proteins present in the UF perform during early pregnancy, a period marked by embryonic implantation, and maternal recognition of pregnancy is of fundamental importance to understanding the mechanisms necessary for the maintenance of pregnancy. The present study characterized and compared the UF proteome at the beginning of pregnancy in pregnant and non-pregnant buffaloes to correlate the functions of the proteins and the stage of development of the conceptus and unravel their processes in maternal recognition of pregnancy. The proteins found were involved in cell growth and endometrial remodeling, in addition to acting in the immunological protection of the conceptus and performing antioxidant actions necessary for establishing a pregnancy.

Proteomics approach reveals urinary markers for early pregnancy diagnosis in buffaloes.

This study aimed to compare urine proteomics from non- and pregnant buffaloes in order to identify potential biomarkers of early pregnancy. Forty-four females underwent hormonal ovulation synchronization and were randomly divided into two experimental groups: inseminated (n = 30) and non-inseminated (n = 14). The pregnant females were further divided into two groups: pregnant at Day 12 (P12; n = 8) and at Day 18 (P18; n = 8) post-ovulation. The non-pregnant group was also subdivided into two groups: non-pregnant at Day 12 (NP12; n = 7) and at Day 18 (NP18; n = 7). Urine was collected from all females on Days 12 or 18. The samples were processed for proteomics. A total of 798 proteins were reported in the urine considering all groups. The differential proteins play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that some proteins from our study can be considered biomarkers for early pregnancy diagnosis, since they were increased in pregnant buffaloes. SIGNIFICANCE: Macromolecules have been studied for early pregnancy diagnosis, aiming to increase reproductive efficiency in cattle and buffaloes. Direct methods such as rectal palpation and ultrasonography have been considered late. Thus, this study aimed to compare urine proteomics from non- and pregnant buffaloes to identify potential biomarkers of early pregnancy. The differential proteins found in our study play essential roles during pregnancy, acting in cellular transport and metabolism, endometrial remodeling, embryonic protection, and degradation of defective proteins. We suggest that these proteins can be considered possible biomarkers for early pregnancy diagnosis since they were increased in the pregnant buffaloes.

Influence of culture conditions on the secretome of mesenchymal stem cells derived from feline adipose tissue: Proteomics approach.

This study aimed to describe the secretome of mesenchymal stem cells derived from feline adipose tissue (AD-MSCs) and compare the effects of different culture conditions on AD-MSC proteomics using a shotgun approach. Adipose tissue was collected from 5 female cats and prepared to culture. Conditioned media was collected at third passage, in which the cells were cultured under 4 conditions, normoxia with fetal bovine serum (N + FBS), hypoxia with FBS (H + FBS), normoxia without FBS (N - FBS), and hypoxia without FBS (H - FBS). Then, the secretome was concentrated and prepared for proteomic approaches. Secretomes cultured with FBS-free medium had more than twice identified proteins in comparison with the secretomes cultured with FBS. In contrast, hypoxic conditions did not increase protein amount and affected only a small proteome fraction. Relevant proteins were related to the extracellular matrix promoting environmental modulation, influencing cell signaling pathways, and providing a suitable environment for cell proliferation and maintenance. Moreover, other proteins were also related to cell adhesion, migration and morphogenesis. Culture conditions can influence protein abundance in AD-MSC secretome, and can give also more specificity to cell and cell-free treatments for different diseases.

Can blood progesterone concentration identify non-pregnant buffaloes to support oestrous resynchronization?

This study compared the plasma progesterone concentrations from pregnant and non-pregnant buffaloes to identify non-pregnant females and submit cows earlier to oestrous resynchronization. Forty-four multiparous mix-breed Murrah buffaloes were selected for the study. The cows were subjected to hormonal oestrous synchronization and separated into 4 groups, P12 (pregnant, n = 8) and P18 (n = 8) at 12 and 18 days post-insemination; NP12 (non-pregnant, n = 7) and NP18 (n = 7) at 23 and 29 days after the onset of synchronization, respectively. The embryos and blood were collected, and the plasma was separated for centrifugation and used to determine progesterone concentration. Progesterone concentration was higher in P18 than P12 (p = .02) and NP18 groups (p < .001). The steroid was also increased in the P12 group compared with NP12 (p = .031). There was no difference between NP12 and NP18 (p = .906). We conclude that the plasma progesterone concentration can be an alternative to identify earlier non-pregnant buffaloes, advancing the oestrous resynchronization or natural service to improve productivity.

Insights into the influence of canine breed on proteomics of the spermatozoa and seminal plasma.

This study aimed to characterize the proteome of spermatozoa and seminal plasma of 4 purebred dogs (Golden Retriever, Great Dane, Bernese Mountain Dog, and Maremmano-Abruzzese Sheepdog). The ejaculate of 13 dogs was collected, and sperm characteristics were subjectively evaluated. Seminal plasma and sperm cells were separated and prepared individually for mass spectrometry. Data were evaluated by univariate and multivariate statistical analysis. A total of 162 proteins were identified, 47 in spermatozoa, 109 in seminal plasma, and 6 in both samples. Serum albumin in spermatozoa and tubulin alpha-3E chain, acrosin binding protein, and tubulin alpha-3 chain in plasma seminal were statistically relevant. Serum albumin and acrosin binding protein improve the sperm capacitation, acrosome reaction, and seminal quality. The tubulin family proteins are related to structural cell organization and flagella movement, and their presence in seminal plasma may be related to sample handling. According to cluster formation, a high association was observed among Bernese Mountain Dog and Great Dane, Golden Retriever, and Maremmano-Abruzzese Sheepdog for sperm proteins. For seminal plasma proteins, Bernese Mountain Dog, Great Dane, and Maremmano-Abruzzese Sheepdog were related. Further studies on breed-specific proteins in the semen of purebred dogs need to be performed to clarify its fertility roles. SIGNIFICANCE: For the first time spermatozoa proteins of dogs are described. The comparison of spermatozoa and seminal plasma proteins of four purebred dogs were performed. These results supporting that differences in semen protein profile of different canine breeds exist, which can improve the biotechnologies of reproduction in this species.

Normal echocardiographic and radiographic reference values for crab-eating fox (Cerdocyon thous) anesthetized with ketamine and midazolam.

The cardiac evaluation of wild animals is still a wide and largely unknown field for several species. Therefore, through complimentary examinations such as radiography, echocardiography and serum troponin levels, this study aimed at describing the values observed in 12 crab-eating foxes (Cerdocyon thous) anesthetized with a combination of intramuscular ketamine and midazolam. Thus, through complementary exams such as radiography, echocardiography and serum troponin levels, the aim of this study was to describe the cardiac values in 12 wild foxes (C. thous) anesthetized with an intramuscular injection of ketamine and midazolam. After anaesthetization, the radiographic, echocardiographic and immunoenzymatic reference values for the 12 males in the sample group were determined. Compared with those in domestic canids, there was a decrease in the sizes of the septum, wall and left ventricular cavity as well as decreases in the transmitral blood flow velocity indices, correlated with preserved serum cardiac troponin (cTnI) levels. Thus, M-mode echocardiography proved to be safer, with results that were comparative to those for other species of wild canids with indexed values. In addition, when evaluating the systolic function and segmentary contractions, the anesthetic combination did not have any effects on the results of complementary examinations performed in crab-eating foxes (C. thous) included in this study.

A comparison of immunological, chemical and surgical castration of Nelore bulls.

The objective was to compare effects of immunological, chemical and surgical castrations on testicular characteristics, scrotal surface temperatures, sperm quality, and serum testosterone concentrations in Bos indicus bulls. Eighty Nelore bulls (∼20 mo) were grazed on pasture, fed a supplement and slaughtered at ∼480 kg. Bulls were allocated into four groups (n = 20/group). The control group (CON) was non-castrated and there were three castration methods: immune (IMM - Bopriva®), injected on D-30, D30 and D60; chemical (CHE), an intratesticular injection of 40% CaCl2 + 0.5% dimethylsulfoxide on D0; and surgical (SUR) on D0. The CHE group were surgically castrated on D60, due to testicular swelling and necrosis of scrotal skin. Most scrotal surface temperatures (infrared thermography) were significantly higher on D15 in CHE and SUR compared to CON, due to inflammation. All bulls were subjected to a breeding soundness evaluation on D-7 and slaughtered on D220. Scrotal circumference and testicular volume did not differ among groups (P > 0.05) at D0, but at D15, both end points were highest (P < 0.05) in the CHE group (due to swelling), although both end points were smaller for IMM versus CON (P < 0.05) at D60 and D150. Sperm motility in the IMM group was ∼50 and 10% of that in the CON group on D60 and D150, respectively. For the IMM group, serum testosterone concentrations were similar on D0 and D15 (but ∼50% of CHE or SUR on D0, attributed to the first treatment on D-30), and had decreased ∼70% on D60 and D150, whereas in the CHE and SUR groups, there were ∼80-90% decreases in testosterone after D0. In conclusion, immunological castration was a viable alternative to surgical castration, as it supressed testosterone production and spermatogenesis, with the benefits of being much less invasive, with better animal welfare and less stress.

Proteomic data of seminal plasma and spermatozoa of four purebred dogs.

Semen contains several proteins that are important to fertilization and to identify reproductive failures. There are proteins that are specie-specific expressed, although differs among several breeds. This article provides experimental data describing the protein profile of seminal plasma and spermatozoa of four healthy purebred dogs: Golden Retriever (n=3), Bernese Mountain Dog (n=4), Great Dane (n=3), and Maremmano-Abruzzese Sheepdog (n=3), housed at São Paulo state, Brazil. Semen samples were collected by manual stimulation of the penis in a presence of a teaser bitch, when possible. The seminal plasma and sperm cells were separated by centrifugation and prepared for mass spectrometry. The gene ontology annotation of the proteins found is described. This is the first time that proteomic profile of the semen of purebred dogs is described. These data are a valuable resource to improve the biotechnologies of reproduction applied to canid species.

Cross comparison of seminal plasma proteins from cattle and buffalo (Bubalus bubalis).

The objective of this study was to evaluate seminal plasma proteins from cattle and buffalo (Bubalus bubalis), to identify differences between related species. Sixteen buffaloes and 16 cattle between 30 and 60 months of age were used. Semen collection was performed by electroejaculation, followed by macroscopic and microscopic subjective analyses. After analysis, the samples were centrifuged at 800 g for 10 min, and the supernatant (seminal plasma) was recentrifuged at 10,000 g for 30 min at 4°C. The total protein concentration was determined by the Bradford method, and the proteins were digested in solution for mass spectrometry (nLC-MS/MS). Multivariate statistical analysis was used to evaluate the proteomics results by non-hierarchical clustering the considering exponentially modified protein abundance index (emPAI). Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) were used for clustering. Proteomics identified 78 proteins, and multivariate analysis showed 4 that were over-expressed in buffaloes (cystatin C, prosaposin, peptide YY and keratin type II cytoskeletal 5) and 9 in cattle (spermadhesin-1, seminal plasma protein PDC-109, ribonuclease 4, metalloproteinase inhibitor 2, acrosin inhibitor 1, seminal ribonuclease, C-type natriuretic peptide, angiogenin-1 and osteopontin). Among the proteins identified in seminal plasma, the C-type natriuretic peptide and metalloproteinase inhibitors were described for the first time in buffaloes. Some protease inhibitors were found over-expressed in buffaloes, and important proteins in seminal plasma of cattle were not identified or were found at lower expression levels in buffaloes, which can contribute to reproductive performance in this species.

Uterine intussusception in immediate postpartum in bitches: case report / Intussuscepção uterina no puerpério imediato em cadelas: relato de caso

Uterine intussusception is a rare condition in bitches. This study aimed to report an unusual case of prolapsed uterine intussusception in a female dog. A 2-year-old American Bully bitch was received with labor history of large fetus requiring manual traction. The animal presented mucosal prolapse through the vulva, unproductive contractions and abdominal discomfort. After partial correction of mucosal prolapse, the dog was referred to ovary-hysterectomy surgery to correct prolapsed mucosa. During the surgical procedure, surgeons observed that the uterine horns were invaginated into the uterine body, rotated, and the tissue was slightly devitalized and congested. As previously described, this condition occurred during the immediate postpartum period. Thus, we suggest that this period can be a facilitator for the establishment of uterine intussusception.(AU)

A intussuscepção uterina é uma condição rara em cadelas. Portanto, este estudo tem por objetivo relatar um caso incomum de intussuscepção uterina prolapsada em fêmea canina. Uma cadela American Bully de dois anos de idade com histórico de parto de feto absoluto grande no qual necessitou de auxílio por tração manual foi apresentada. O animal exibiu um prolapso de mucosa através da vulva, contrações improdutivas e dores abdominais. Após a retração parcial do prolapso de mucosa, a cadela foi encaminhada para a ovário-histerectomia que teve por objetivo, também, corrigir a mucosa prolapsada. Durante o procedimento cirúrgico, foram verificados os cornos uterinos invaginados no corpo uterino, rotacionados, com o tecido levemente desvitalizado e congesto. Assim, como poucos casos encontrados na literatura, esta condição ocorreu durante o puerpério imediato. Dessa forma, sugere-se que este período, embora não seja um fator determinante para o estabelecimento da intussuscepção uterina, pode ser um facilitador no estabelecimento dessa condição.(AU)

Proteomic analysis of amniotic and allantoic fluid from buffaloes during foetal development.

The objective of this study was to describe the dynamic changes in protein composition and protein abundance in amniotic and allantoic fluids from buffaloes during gestation. Amniotic and allantoic fluids were collected during the first, second and third trimesters of gestation. The foetuses were measured and weighed. Fluid samples were centrifuged at 800 g for 10 min and then at 10,000 g for 60 min at 4°C. The supernatant was collected to determine the total protein concentration. Based on total protein concentration, an aliquot (50 µg) was used for in-solution tryptic digestion, and mass spectrometry analysis (nano-LC-MS/MS) was performed. A multivariate statistical analysis of the proteomic data was conducted. Across the different stages of buffalo gestation, fifty-one proteins were found in the amniotic fluid, and twenty-one were found in the allantoic fluid. A total of twelve proteins were common among the stages, and four presented significant differences (VIP score α > 1). Fibronectin and alpha-1-antiproteinase were more abundant in the amniotic fluid than in the allantoic fluid. Alpha-2-macroglobulin and alpha-2-HS-glycoprotein were more abundant in the allantoic fluid than in the amniotic fluid. Alpha-2-macroglobulin participates in remodelling and growth of the uterus at beginning of the gestation (first trimester), and these findings indicate that can serve as a potential tool for the early diagnosis of pregnancy in buffaloes.

Effect of estrous cycle phase on vulvar, orbital area and muzzle surface temperatures as determined using digital infrared thermography in buffalo.

The objective of this study was to evaluate variations in the orbital area, muzzle and vulva surface temperatures and progesterone (P4) concentrations during follicular and luteal phases in Murrah buffalo and whether these temperatures are influenced by the weather patterns. Forty cows were submitted to P4-based hormonal protocol. After P4 device withdrawal transrectal ultrasonography and infrared digital thermography were performed daily until day 16 and on days 20, 24, 28 and 32 to follow the ovulation as well as the vulva, orbital area and muzzle temperatures. In addition, the weather variables were evaluated, as well as rectal temperature (RT) and P4 and cortisol concentration. Vulva, muzzle and orbital area temperatures correlated positively with RT and with weather data. Greater temperatures of the vulva, orbital area and muzzle were detected during the period of estrus. The vulvar surface temperature (VST) was not influenced to a great extent by weather factors during the morning, so this period was chosen to evaluate the influence of the phase of the estrous cycle on VST. The VST was less during days 16, 20, 24 and 28 (diestrus) and P4 concentration was inversely proportional to the VST. Muzzle, orbital area and RT, however, were not of the same pattern. Negative correlations were observed between VST and P4 concentrations. It is concluded that VST undergoes changes during the reproductive phases, correlating with P4 concentration. The weather factors influence the temperatures of the body surface areas, and the morning is the most desirable time to perform the thermographies.

Functional insights into the role of seminal plasma proteins on sperm motility of buffalo.

The objective of the present study was to describe the proteins from the seminal plasma of buffalo and correlate these proteins with sperm motility. Ejaculates from sixteen Murrah buffalo were used. Semen collection was performed by electroejaculation, and the ejaculate was evaluated by macroscopic (volume) and microscopic analysis (subjective motility and vigor, as well as sperm concentration). After the analysis, the samples were centrifuged (800g for 10 min and 10,000 for 30 min at 4 °C), and the supernatant (seminal plasma) was used to determine total protein concentration by the Bradford method. Based on total protein concentration, an aliquot (50 µg) was taken to conduct protein in-solution digestion for nano-LC-ESI-Q-TOF mass spectrometry analysis. Samples were divided into two groups, minimal (little sperm motility) and greater (typical sperm motility), based on non-hierarchical clustering considering motility and emPAI protein value. The data were analyzed by multivariate statistical analysis using principal component analysis (PCA) and partial analysis of minimum squares discrimination (PLS-DA). Forty-eight proteins were detected in the seminal plasma, and fifteen were common to two groups. There were six proteins that were significantly different between the groups. The main functions of proteins in seminal plasma were catalytic and binding activity. Spermadhesin protein, ribonuclease, 14-3-3 protein zeta/delta and acrosin inhibitor were in greater amounts in seminal plasma from the group with greater sperm motility; prosaposin and peptide YY were in greater amounts in the group with little sperm motility. The proteins detected in the greater motility group were correlated with sperm protection, including protection against oxidative stress, lipid peroxidation, protease inhibition and prevention of premature capacitation and acrosome reaction. In the group with little sperm motility, one of the identified proteins is considered to be an antifertility factor, whereas the function of other identified protein is not definitive. Results from the present study add to the knowledge base about the molecular processes related with sperm motility, and these findings can be used for determining potential markers of semen quality.

Evaluation of buffaloes' follicular dynamics and stress state under different ovulation synchronization protocols.

The aims of the present study were to analyze the effect of different hormonal protocols using an implant containing Norgestomet, at the ovulation synchronization in buffaloes and to verify the effect of the stress caused by manipulation of the herd during the experiment. Twenty-four female Murrah breed buffaloes, lactating, multiparous, aged from three to eight years, with a body condition score of 3.5 or higher (0 to 5) and with more than 45 days post partum, were used. These females were randomly distributed into one of the three groups (group 1, group 2 and group 3) with eight subjects in each. On day 0 (day 0), all animals received an ear progesterone implant with 3.0 mg of Norgestomet and an intramuscular (IM) injection with 2.0 mg of estradiol benzoate (EB). The implants were removed after nine days (day 9) and one single dose of PGF2α (0.15 mg d-cloprostenol, IM) was administered to all animals. On the same day, the group´1 and group 3 buffaloes were treated with 500 UI of eCG (IM). Two days later (day 11), 1000UI of hCG (IM) were administered to the group 1 and group 2 buffaloes. After the implant had been removed, an ultrasound evaluation was performed every 12 h, in order to access the ovarian follicular dynamics, using an Aloka 500 equipment with a 5 MHz transrectal probe. Blood samples were also taken on days 0, 9 and 11 to determine the plasmatic concentrations of cortisol and progesterone. No difference was observed in the ovulation rate (group 1: 62.5%, group 2: 50%, group 3: 75%). However, the size of the preovulatory follicles and the plasmatic concentration of cortisol had (P < 0.05). G3 was the most efficient group in promoting the ovulation synchronization, which suggests that this protocol can be used in Fixed Timed Artificial Insemination programs (FTAI) among postpartum, lactating Murrah breed buffaloes'.