Aquaporins in the male reproductive tract and sperm: Functional implications and cryobiology.

Aquaporins (AQPs) play a vital role for the transport of water and solutes across cell membranes. Classification of these ubiquitous proteins into three categories (orthodox AQPs, aquaglyceroporins and superaquaporins) is based on their sequence similarity and substrate selectivity. In the male reproductive tract of mammals, most AQPs (except AQP6 and AQP12) are found in different organs (including testis, efferent ducts and epididymis). AQP1 and AQP9 are the most abundant AQPs in the efferent ducts and epididymis and play a crucial role for the secretion/reabsorption dynamics of luminal fluid during sperm transport and maturation. AQP3, AQP7, AQP8 and AQP11 are the most abundant AQPs in sperm and are involved in the regulation of their volume, which is required for the differentiation of spermatids into spermatozoa during spermatogenesis, as well as in sperm transit along environments of different osmolality (male and female reproductive tracts). While different studies conducted in oocytes and embryos have demonstrated that AQPs are important for cryotolerance, data in sperm are scarce. At present, mounting evidence indicates that AQP3, AQP7 and AQP11 are involved in the sperm response to variations of osmolality and to freeze-thawing procedures. All these studies contribute to understand the physiology of both male reproductive tract and sperm, and open up new research ventures on the improvement of sperm cryopreservation protocols.

Cryotolerance of in vitro-produced porcine blastocysts is improved when using glucose instead of pyruvate and lactate during the first 2 days of embryo culture.

The objective of the present study was to determine the effects of replacing glucose with pyruvate and lactate during the first 48 h of in vitro culture (IVC) in NCSU-23 medium on embryo development, embryo quality and survival of porcine blastocysts after vitrification. To this end, in vitro-produced (IVP) porcine oocytes were cultured with either glucose for 6 days (IVC-Glu) or pyruvate-lactate from Day 0 to Day 2 and then with glucose until Day 6 (IVC-PyrLac). Blastocysts were vitrified on Day 6 using the Cryotop device and, after warming, survival rate and the apoptosis index were evaluated after 24 h incubation in NCSU-23 medium. No significant differences were observed between IVC-Glu and IVC-PyrLac in terms of cleavage rate, blastocyst yield, total number of cells per blastocyst or the apoptosis index (1.82±0.75% vs 3.18±0.88%, respectively) of non-vitrified embryos. However, a significant increase was seen in hatching/hatched blastocysts in the IVC-PyrLac compared with IVC-Glu treatment group (12.71±1.20% vs 3.54±0.47%, respectively). Regardless of treatment, vitrification impaired the survival rate and the apoptosis index. When comparing both treatments after warming, the percentage of apoptotic cells was significantly higher for blastocysts in the IVC-PyrLac compared with IVC-Glu group (18.55±3.49% vs 9.12±2.17%, respectively). In conclusion, under the conditions of the present study, replacement of glucose with pyruvate-lactate during the first 48 h of culture resulted in a lower cryotolerance of IVP porcine embryos.

Assessment of meiotic spindle configuration and post-warming bovine oocyte viability using polarized light microscopy.

The objectives of this study were to assess the efficiency of polarized light microscopy (PLM) in detecting microtubule-polymerized protein in in vitro-matured bovine oocytes; to examine its effects on oocyte developmental competence; and to assess the meiotic spindle of in vitro-matured oocytes after vitrification/warming and further assessment of oocyte developmental competence. In the first experiment, the presence of microtubule-polymerized protein (MPP) was confirmed as a positive PLM signal detected in 99.1% of analysed oocytes (n = 115), which strongly correlated (r = 1; p < 0.0001) with the presence of MPP as confirmed by immunostaining. In the second experiment, oocytes (n = 651) were exposed or not (controls) to PLM for 10 min and then fertilized and cultured in vitro. Oocytes exposed to PLM did not significantly differ from controls with regard to cleavage, total blastocyst and expanded blastocyst rates and cell numbers. In the third experiment, meiotic spindles were detected in 145 of 182 oocytes (79.6%) following vitrification and warming. Interestingly, after parthenogenetic activation and in vitro culture, oocytes that displayed a positive PLM signal PLM(+) differed significantly from PLM(-) in cleavage and Day 8 blastocyst rates. These results suggest that polarized light microscopy is an efficient system to detect microtubule-polymerized protein in in vitro-matured bovine oocytes and does not exert detrimental effects on bovine oocyte developmental competence. Moreover, PLM could be used as a tool to assess post-warming viability in vitrified bovine oocytes.

Developmental competence and embryo quality of small oocytes from pre-pubertal goats cultured in IVM medium supplemented with low level of hormones, insulin-transferrin-selenium and ascorbic acid.

The aim of this study was to test the effect of insulin-transferrin-selenium (ITS) and L-ascorbic acid (AA) supplementation and the hormonal level during in vitro maturation (IVM) of small oocytes from pre-pubertal goat on the blastocyst yield and quality. Concretely, we used four maturation media: conventional IVM medium (CM), growth medium (GM: CM+ITS+AA and low level of hormones), modified CM (mCM: CM with low level of hormones) and modified GM (mGM: CM+ITS+AA and normal level of hormones). Cumulus-oocyte complexes (COCs) were classified into two categories according to oocyte diameter: <125 µm and ≥ 125 µm. Large oocytes were matured 24 h in CM (Treatment A). Small oocytes were matured randomly in six experimental groups: Treatment B: 24 h in CM; Treatment C: 12 h in GM and 12 h in CM; Treatment D: 24 h in mGM; Treatment E: 12 h in mGM and 12 h in CM; Treatment F: 12 h in mCM and 12 h in CM; and Treatment G: 12 h in GM and 12 h in mGM. After IVM, oocytes were fertilized and cultured for 8 days. The blastocyst quality was assessed by the survival following vitrification/warming and the mean cell number. When different maturation media were combined, the blastocyst rate did not improve. The large oocytes produced the highest blastocysts yield. However, the culture of small oocytes in GM (53.3%) enhanced the post-warming survival of blastocysts compared to large oocytes matured in CM (35.7%). In conclusion, IVM of pre-pubertal goat small oocytes in GM would be useful to improve the quality of in vitro-produced blastocysts.

The effect of habitat fragmentation on the genetic structure of a top predator: loss of diversity and high differentiation among remnant populations of Atlantic Forest jaguars (Panthera onca).

Habitat fragmentation may disrupt original patterns of gene flow and lead to drift-induced differentiation among local population units. Top predators such as the jaguar may be particularly susceptible to this effect, given their low population densities, leading to small effective sizes in local fragments. On the other hand, the jaguar's high dispersal capabilities and relatively long generation time might counteract this process, slowing the effect of drift on local populations over the time frame of decades or centuries. In this study, we have addressed this issue by investigating the genetic structure of jaguars in a recently fragmented Atlantic Forest region, aiming to test whether loss of diversity and differentiation among local populations are detectable, and whether they can be attributed to the recent effect of drift. We used 13 microsatellite loci to characterize the genetic diversity present in four remnant populations, and observed marked differentiation among them, with evidence of recent allelic loss in local areas. Although some migrant and admixed individuals were identified, our results indicate that recent large-scale habitat removal and fragmentation among these areas has been sufficiently strong to promote differentiation induced by drift and loss of alleles at each site. Low estimated effective sizes supported the inference that genetic drift could have caused this effect within a short time frame. These results indicate that jaguars' ability to effectively disperse across the human-dominated landscapes that separate the fragments is currently very limited, and that each fragment contains a small, isolated population that is already suffering from the effects of genetic drift.

One-step warming does not affect the in vitro viability and cryosurvival of cryotop-vitrified donkey embryos.

The objective of this study was to compare the effects of two warming protocols (three-step vs. one-step dilution) on embryo quality, post-warming embryo survival and embryo cell viability of donkey embryos vitrified by the Cryotop method. Twenty, Day 7-8, grade 1-2 donkey embryos were measured, morphologically evaluated and vitrified using the Cryotop technique. Embryos were then randomly warmed using two different warming procedures: (i) W3 (three-step dilution; n = 11): embryos were warmed in 1 M, 0.5 M and 0 M sucrose, and (ii) W1/0.5 (one-step dilution; n = 9): embryos were warmed directly in 0.5 M sucrose. After 3 and 24 h of warming, the embryos were measured and evaluated for their morphology, developmental stage and viability (Propidium Iodide-Hoechst 33,342 dyes). Although both treatments decreased embryo quality after warming (P < 0.05), no significant differences (P > 0.05) were observed between protocols in terms of post-warming embryo quality, diameter and embryo survival. Greater percentages of dead cells (P < 0.001) were observed when embryos were warmed directly in 0.5 M sucrose (one-step dilution) when compared to the three-step protocol. The percentage of ruptured embryos was 27.3% and 0% in W3 and W1/0.5 protocols (P = 0.0893), respectively. In conclusion, warming Cryotop-vitrified donkey embryos directly in 0.5 M sucrose had no negative effects on embryo quality and post-warming embryo survival. Moreover, one-step protocol may help to prevent rupture when donkey embryos warmed directly in 0.5 M sucrose. These results observed in vitro must be verified by embryo transfer.

The cryoprotective effect of Ficoll 70 on the post-warming survival and quality of Cryotop-vitrified donkey embryos.

Many domestic donkey breeds are at risk of extinction, there is a critical urgency for genome resource banking. In the present study, we examined whether the use of Ficoll 70 added to the vitrification medium containing ethylene glycol (EG), dimethyl sulfoxide (DMSO) and sucrose improves the cryotolerance of donkey in vivo derived embryos. Day 7-8, grade 1-2 donkey embryos were measured and morphologically evaluated and then vitrified-warmed using the Cryotop technique. Before vitrification, embryos were randomly distributed into two groups: (i) VS1 (n = 14): vitrified using 15% EG + 15% DMSO + 0.5 M sucrose; and (ii) VS2 (n = 10): vitrified in the same medium supplemented also with 18% of Ficoll 70. After 24 h of warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). Post-warming survival was numerically higher but not significantly different (P > 0.05) when embryos were vitrified in VS2 (70%) compared to VS1 (57.1%). Embryo rupture was only observed in the VS1 group (21.4%, 3/14). Higher embryo diameter was observed in all groups after 24 h culture (P < 0.05). No significant differences (P > 0.05) were observed among treatments in terms of percentages of cell death. These results demonstrate that the addition of Ficoll 70 to the vitrification medium is not a pre-requisite for successful vitrification of donkey embryos. However, its addition seems to enhance some of the post-warming embryo quality characteristics. Since no statistically significant evidence was found, further studies should be conducted in order to confirm our findings.

Hematology and blood chemistry parameters differ in free-ranging maned wolves (Chrysocyon brachyurus) living in the Serra da Canastra National Park versus adjacent farmlands, Brazil.

There has been growing interest in the specific impacts of anthropogenic factors on the health of wildlife. This study examined hematology and serum chemistry status of a prominent carnivore, the maned wolf (Chrysocyon brachyurus), living in, on the boundaries to, or on adjacent farmlands to the Serra da Canastra National Park, Brazil. Twenty-eighty wolves were captured, and values were compared 1) between subadults (n=8 animals) and adults (n=20 animals), 2) males (n=12 animals) and females (n=16 animals), and 3) among wolves living inside the park (n=11), near the park border (n=11 animals), and in neighboring farming areas (n=6 animals). Age, gender, and wolf locations influenced (P<0.05) hematology and serum biochemistry values. Specifically, adults had lower (P<0.05) circulating phosphorus than subadults. Males had lower (P<0.05) serum glucose, creatinine phosphokinase, and cholesterol and higher (P<0.05) potassium than females. Erythrocyte count and serum cholinesterase were lower (P<0.05) in wolves living within the park compared with near the park border or on farmlands. Mean corpuscular volume was lower (P<0.05) in wolves living near the park border than those ranging within the park and on farmlands. Aspartate transaminase and chloride were higher (P<0.05) in wolves living inside the park compared with those ranging near the park border. Creatinine phosphokinase was lower (P<0.05) in wolves living on farmland compared with the other two locations. These results clearly reveal a relationship between age and gender on hematology and serum biochemistry values in free-living maned wolves. More importantly, certain traits indicative of health are potentially compromised in wolves living in areas under anthropogenic pressure. These data lay a foundation for examining the influence of farming and local domestic species on disease susceptibility and fitness in the maned wolf.

Cryopreservation of donkey embryos by the cryotop method: Effect of developmental stage, embryo quality, diameter and age of embryos.

Cryopreservation of embryos has the potential to become a valuable tool for the conservation of endangered donkey breeds. However, there are several factors that can affect cryosurvival of embryos. This study evaluates the effectiveness of the Cryotop method to vitrify donkey embryos and factors affecting the survival of vitrified-warmed embryos. Day 6-8 embryos were measured and morphologically evaluated. Embryos were then vitrified-warmed using the Cryotop technique. After 24 h post-warming, the embryos were measured and evaluated for their morphology, development and viability (Propidium Iodide-Hoechst 33342 dyes). A total of 25 embryos were used, of which 17 were classified as Grade 1 (excellent), 5 as Grade 2 (good) and 3 as Grade 3 (fair). Based on their diameter, embryos were grouped as follows: ≤220 µm (n = 10), >220-300 µm (n = 8), and >300 µm (n = 7). Post-warming survival of vitrified embryos was similar (P > 0.05) to the control fresh embryos, regardless of embryonic diameter, developmental stage, and age of the embryos before vitrification. However, the proportion of embryos that survived vitrification procedures was numerically higher but not significantly different (P > 0.05) for Day 7 embryos (84.6%). The ability of Grade 1 (70.6%) and 2 (80%) embryos to survive vitrification procedures was higher (P = 0.0214) than those of Grade 3 (0%). The proportion of dead cells in Grade 3 embryos (56.5%) was higher (P < 0.01) than that of Grade 1 (3.2%) and 2 (1.5%) embryos. In conclusion, the Cryotop technique seems to be useful for Grade 1 and 2 donkey embryos. It is likely that donkey embryos show similar survival rates after vitrification in Cryotops irrespective of age, diameter and development stage.

Patient satisfaction in a Spanish burn unit.

BACKGROUND: As a result of the Spanish healthcare system overhaul, quality of care is becoming increasingly important. All burn service providers are required to measure patient satisfaction with care as an imperative need. Nevertheless, there are very few papers regarding patient satisfaction in burn units or in plastic surgery in general. The aim of this study is to examine patient satisfaction in our burn unit and to identify areas for improvement. MATERIALS AND METHODS: Participants were all patients admitted to the Burn Unit at the Getafe University Hospital (Madrid, Spain) between January 2014 and December 2016. Patient satisfaction was assessed using the SERVQHOS questionnaire and Kano methodology. The SERVQHOS questionnaire was given to all patients at the time of discharge with completion thereof voluntary and anonymous. The Kano model consisted of an in-depth personal interview with patients and their relatives to identify patient requirements. Further, we developed a Kano questionnaire and analysed the results to prioritise the requirements for development activities. RESULTS: A total of 164 SERVQHOS questionnaires were collected, which means 58% of the discharged patients who were asked to participate returned the questionnaire. Mean overall satisfaction score was 3.7 (range 1-4). Ninety-seven per cent of patients would not hesitate to recommend the hospital to others, 90% believed they had stayed in the hospital for the time necessary and 89% did not have any pain relief problems. The issues that were rated the worst by users were those related to objective quality such as room conditions, location directions, ease of discharge from the hospital and employee appearance. The best-valued aspects were those related to subjective quality such as willingness to help patients, ability to inspire trust and confidence, courtesy and personal attention. CONCLUSIONS: Patients hospitalised in our burn unit are highly satisfied with the care they receive, especially with regard to subjective quality. The evaluation of the satisfaction outcomes helped us to identify several strengths and weaknesses in the healthcare services we provide as well as strategies to improve the weaknesses. Evaluating care quality and patient satisfaction in any burn unit is appropriate and recommendable given that it offers clients' first-hand opinions.

Embryo development and structural analysis of in vitro matured bovine oocytes vitrified in flexipet denuding pipettes.

The aim of this study was to compare the effectiveness of two different vitrification carrier systems for oocyte cryopreservation. In vitro matured (IVM) bovine oocytes were vitrified in open pulled straws (OPS) or flexipet denuding pipettes (FDP), and the effects of cryopreservation determined on the cytoskeletal components and developmental capacity of the oocytes. Three experimental groups were established according to whether the oocytes were vitrified in OPS (OPS group), FDP (FDP group) or left untreated (CTR group). Twenty two hours after the onset of maturation, sub-groups of 2-4 oocytes were pre-equilibrated in 1 mL of Hepes-TCM 199 with 20% fetal calf serum (FCS) (HM), 10% dimethyl sulfoxide (DMSO) and 10% ethylene glycol (EG) for 30 s. The oocytes were then transferred to a 20-microL drop of HM containing 20% DMSO, 20% EG and 0.5 M of sucrose, which was used to load the OPS or FDP before their immersion in liquid nitrogen (LN2). Oocytes were thawed by plunging the OPS or FDP into 0.25 M sucrose in HM, and then placed for 5 min each in 0.15 and 0 M sucrose in HM. After warming, spindle configuration, chromosome distribution and embryo development were assessed. Frozen-thawed semen was used for fertilization. Zygotes were denuded at 22 h post-insemination, and cultured in SOF medium for 9 days at 38.5 degrees C in a 5% CO2, 5% O2 and 90% N2 atmosphere. All experiments were performed using both cow and calf oocytes to establish sensitivity differences. After in vitro fertilization and culture, oocytes in the FDP group showed a lower cleavage rate than those in the OPS or control groups (P<0.05), while blastocysts were only obtained in the OPS group, at a lower rate than controls. After warming, double fluorescent staining revealed higher rates of spindle and chromosome abnormalities in the FDP group compared to the OPS group (P<0.05). No differences between cow and calf oocytes were observed in the different experiments. Our results indicate that the carrier system affects the capacity of IVM oocytes to survive cryopreservation. Unexpectedly, the flexipet denuding pipette failed to improve results and high rates of clustered chromatin and abnormal spindles were observed in calf and cow oocytes vitrified by the FDP method. In conclusion, the use of the flexipet denuding pipette modifies the cytoskeletal components and compromises the developmental capacity of in vitro matured calf and cow oocytes.

Duration of spermatogenesis and daily sperm production in the jaguar (Panthera onca).

The jaguar, like most wild felids, is an endangered species. Since there are few data regarding reproductive biology for this species, our main goal was to investigate basic aspects of the testis and spermatogenesis. Four adult male jaguars were utilized; to determine the duration of spermatogenesis, two animals received an intratesticular injection of H(3)-thymidine. Mean (+/-SEM) testis weight and the gonadosomatic index were 17.7+/-2.2g and 0.05+/-0.01%, respectively, whereas the seminiferous tubules and the Leydig cells volume density were 74.7+/-3.8 and 16.7+/-1.6%. Eight stages of spermatogenesis were characterized, according to the tubular morphology system and acrosome development. Each spermatogenic cycle and the entire spermatogenic process (based on 4.5 cycles) lasted approximately 12.8+/-0.01 and 57.7+/-0.07 d. The number of Sertoli and Leydig cells per gram of testis was 29+/-4 x 10(6) and 107+/-12 x 10(6). Based on the number of round spermatids per pachytene spermatocyte (2.8+/-0.3:1; meiotic index); significant cell loss (30%) occurred during the two meiotic divisions. There were approximately eight spermatids for each Sertoli cell (Sertoli cell efficiency), whereas the daily sperm production per gram of testis was 16.9+/-1.2 x 10(6). We expect that in the near future, the knowledge obtained in the present investigation will facilitate, utilizing germ cell transplantation, preservation of the germinal epithelium and the ability to generate sperm from jaguars in testes of domestic cats.

Effect of ICSI and embryo biopsy on embryo development and apoptosis according to oocyte diameter in prepubertal goats.

ICSI and embryo biopsy are routine methods used for assisted reproduction. However, their impact on embryo quality is still poor studied. Moreover, oocyte size is also a crucial factor for blastocyst production. In this study effect of oocyte size, ICSI and embryo biopsy was assessed in terms of incidence of apoptosis and blastocyst development. IVM-oocytes from prepubertal goats were fertilized by ICSI or IVF. Embryos obtained were divided depending on oocyte size, biopsied at day-4 post-insemination/injection and cultured for additional 4-5 days. Apoptotic cell number was assessed by TUNEL staining in day-4 embryos and blastocysts obtained. In each diameter group, ICSI did not affect embryo development, blastocyst cell number and embryo apoptotic grade in comparison to IVF. Embryo biopsy did not affect blastocyst rate and apoptotic cell number, but decreased blastocyst cell number (P=0.0018). Moreover, there was a negative relationship between blastocyst cell number and apoptotic grade (P<0.05). In conclusion, ICSI and embryo biopsy do not have negative effect on embryo quality and development. However, oocyte size has a positive relationship on blastocyst yield and quality.

Relationship of aquaporins 3 (AQP3), 7 (AQP7), and 11 (AQP11) with boar sperm resilience to withstand freeze-thawing procedures.

Cryopreservation is the most suitable method to preserve boar spermatozoa over long-term storage. However, freeze-thawing protocols inflict extensive damage to sperm cells, reducing their viability and compromising their fertilizing ability. In addition, high individual variability is known to exist between boar ejaculates, which may be classified as of good (GFE) or poor (PFE) freezability. While conventional spermiogram parameters fail to predict sperm cryotolerance in fresh spermatozoa, high levels of certain proteins, also known as freezability markers, have been found to be related to the sperm resilience to withstand freeze-thawing procedures. In this context, the hypothesis of this study was that aquaporins AQP3, AQP7, and AQP11 could be linked to boar sperm cryotolerance. Twenty-nine ejaculates were evaluated and subsequently classified as GFE or PFE based upon their sperm viability and motility at post-thawing. Fourteen ejaculates resulted to be GFE, whereas the other fifteen were found to be PFE. Relative abundances of AQP3, AQP7, and AQP11 and their localization patterns were evaluated in all fresh and frozen-thawed ejaculates through immunoblotting and immunocytochemistry. Prior to cryopreservation, relative amounts of AQP3 and AQP7 were found to be significantly (p < 0.05) higher in GFE than in PFE. In contrast, no significant differences (p > 0.05) between freezability groups were found for AQP11, despite GFE tending to present higher levels of this protein. The localization of AQP7, but not that of AQP3 or AQP11, was observed to be affected by cryopreservation procedures. In conclusion, these results suggest that AQP3 and AQP7 are related to boar sperm cryotolerance and may be used as freezability markers.

Colheita farmacológica de sêmen de onças-pardas (Puma concolor: Mammalia: Carnivora: Felidae) / Pharmacological semen collection in cougars (Puma concolor: Mammalia: Carnivora: Felidae)

Objetivou-se, por meio do presente estudo, avaliar o método de colheita farmacológica de sêmen com sondagem uretral, em machos de onças-pardas (Puma concolor) mantidos em cativeiro. A técnica proposta (Cat; N=3) foi comparada com a eletroejaculação (EE; N=4). Para a colheita farmacológica, utilizou-se medetomidina para induzir a liberação de sêmen na uretra e sonda uretral para gatos, sem janela lateral, para colheita do sêmen por capilaridade. O método foi eficaz em todos os animais usados. Por meio dessa técnica, colheram-se amostras com menor volume (106,7±30,5aµL) e maior concentração (524,1±54,3b x 106 espermatozoides/mL) em relação à EE (450,0±0,1bµL e 205,0±141,8a x 106 espermatozoides/mL). As avaliações de vigor, motilidade e patologia espermática demonstraram que a técnica não afeta a qualidade do sêmen em relação à EE (P>0,05). Dessa forma, o método proposto consiste em uma técnica mais prática e eficiente para a colheita de sêmen com boa qualidade, dispensando o eletroejaculador.(AU)

The aim of this study was to evaluate the pharmacological semen collection method with urethral catheterization (CT) in captive cougar (Puma concolor) males. The pharmacological method (CT; N= 3) was compared to the electroejaculation technique (EE; N= 4). For CT collection, medetomidine was administrated to induce semen release using a tomcat catheter inserted into the urethra to collect by capillarity. The proposed method was efficacious on all animals used. Through the CT method, semen collected yielded smaller volume (106,7±30,5aµL) and higher concentration (524,1±54,3b x 106sperm/mL) compared to EE (450,0±0,1bµL and 205,0±141,8a x 106 sperm /mL). Evaluations of vigor, motility and sperm pathology demonstrated that CT does not affect semen quality when compared to EE (P> 0.05). Thus, the proposed method consists of a more practical and efficient technique for semen collection with good quality, eliminating the need for eletroejaculation.(AU)

Changes in the fecal concentrations of cortisol and androgen metabolites in captive male jaguars (Panthera onca) in response to stress.

In the present study we determined the efficacy of the measurement of fecal cortisol and androgen metabolite concentrations to monitor adrenal and testicular activity in the jaguar (Panthera onca). Three captive male jaguars were chemically restrained and electroejaculated once or twice within a period of two months. Fecal samples were collected daily for 5 days before and 5 days after the procedure and stored at -20 degrees C until extraction. Variations in the concentrations of cortisol and androgen metabolites before and after the procedure were determined by solid phase cortisol and testosterone radioimmunoassay and feces dry weight was determined by drying at 37 degrees C for 24 h under vacuum. On four occasions, fecal cortisol metabolite levels were elevated above baseline (307.8 +/- 17.5 ng/g dry feces) in the first fecal sample collected after the procedure (100 to 350% above baseline). On one occasion, we did not detect any variation. Mean (+/- SEM) fecal androgen concentration did not change after chemical restraint and electroejaculation (before: 131.1 +/- 26.7, after: 213.7 +/- 43.6 ng/g dry feces). These data show that determination of fecal cortisol and androgen metabolites can be very useful for a noninvasive assessment of animal well-being and as a complement to behavioral, physiological, and pathological studies. It can also be useful for the study of the relationship between adrenal activity and reproductive performance in the jaguar.

In vitro developmental competence of prepubertal goat oocytes cultured with recombinant activin-A.

The present study was designed to evaluate the effect of activin-A during the in vitro oocyte maturation (IVM) and in vitro embryo culture (IVC) on nuclear maturation, blastocyst yield and blastocyst quality of prepubertal goat oocytes. In Experiment 1, three groups of oocytes were used during the IVM of prepubertal goat oocytes to determine the optimal concentration of recombinant human activin-A added to the maturation medium. Cumulus-oocyte complexes were matured in an IVM medium containing 0, 10 and 100 ng/ml (groups A0, A10 and A100), fertilized and in vitro cultured using standard procedures. In Experiment 2, the addition of 10 ng/ml activin-A at IVM (A10A0), IVC (A0A10) or IVM+IVC (A10A10) was studied and compared with the control group (A0A0). Results of the first experiment demonstrated that the addition of activin-A yielded similar percentages of maturation (⩽71.0%) and blastocyst formation rates (⩽24.9%) than the control group (A0). Experiment 2 showed that exposure of prepubertal goat oocytes to an IVC medium containing 10 ng/ml activin-A (A0A10) significantly increased the rates of development to the blastocyst stage, as compared with the control group (A0A0) (19.5±2.21% v. 13.1±2.37%, respectively; P<0.05). With regard to the blastocyst quality, total number of cells, inner cell mass (ICM) and trophectoderm of prepubertal goat embryos produced in the presence of activin-A did not differ significantly among experimental groups. In summary, these results indicate that supplementation of the IVC medium with activin-A enhances embryo development of prepubertal goat oocytes.

Evaluation of sperm subpopulation structure in relation to in vitro sperm-oocyte interaction of frozen-thawed semen from Holstein bulls.

The present study examined the relationship between the relative amount of high motile sperm and sperm-oocyte interactions obtained from Holstein bull ejaculates. Post-thaw sperm motility was analyzed using a computer-assisted sperm analyzer system and evaluated to determine the sperm motility subpopulations. Adhesion and penetration of zona pellucida (ZP) and pronucleus formation using post-thawed samples (15 ejaculates form 5 different bulls) with different percentages of sperm in the subpopulation with the fastest and most progressive subpopulation (subpopulation 4 [SP4]) were analyzed. The correlation between the proportion of sperm in SP4 and the number of spermatozoa bound to the zona pellucida (ZBA), the penetration rate, and the rate of pronucleus formation were calculated. A significant (P < 0.05) and positive correlation was found between the number of spermatozoa bound to the zona pellucida, the penetration rate, and the rate of pronucleus formation with the proportion of sperm in SP4 (r = 0.79, r = 0.66, and r = 0.63, respectively). Our results suggest that this specific high motile and progressive subpopulation is positively and significantly correlated with the ability of a thawed bull semen sample to interact properly with the oocyte and its extracellular vestments. These findings emphasize the relevance of analyzing semen subpopulation composition to predict bull sperm fertilizing ability and to select Holstein bulls for breeding purposes.