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Abstract The aim of this study was to evaluate the effects of in vivo oocyte maturation using seminal plasma alone or in combination with an additional period of in vitro maturation (IVM) on the developmental competence of alpaca oocytes. The experiment was conducted in Lima, Peru, with twelve adult female alpacas. Follicular ablation of the dominant follicle was performed to initiate a new follicular wave. After 36 hours, a superstimulation protocol with 750 IU of eCG was administered intramuscularly (IM). Four days later, 2 mL of seminal plasma was administered IM to promote in vivo oocyte maturation. Cumulus-oocyte complexes (COCs) were retrieved via ovum pick-up 20 hours post-treatment, morphologically evaluated, and allocated into three groups: (T1) no additional IVM, (T2) 12 hours of additional IVM, and (T3) 18 hours of additional IVM. Oocyte developmental competence was assessed using a 26 µM brilliant cresyl blue (BCB) staining protocol for 90 minutes. COCs were classified as BCB positive (blue ooplasm) or BCB negative (unstained ooplasm) and subsequently denuded and fixed for nuclear maturation assessment via orcein staining. No significant differences (p=0.14) were observed in the percentage of expanded COCs across groups (71.4%, 81.8%, and 54.6% for T1, T2, and T3, respectively). The proportion of COCs reaching the metaphase II (MII) stage was higher (p<0.05) in T3 (54.6%), while the developmental competence rate was greatest (p<0.05) in T2 (100%). However, no differences (p=0.21) were detected in the proportion of BCB positive MII stage COCs across groups. In conclusion, alpaca oocytes matured in vivo with seminal plasma require 12 to 18 hours of IVM to achieve optimal nuclear maturation and developmental competence.

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Abstract Luteinizing hormone (LH) plays a crucial role in follicle development, ovulation induction, and the regulation of key reproductive events. However, the efficacy of LH within the follicular microenvironment largely depends on the capacity of follicular cells to express its receptor. This study aims to investigate whether granulosa cells (GCs) can acquire LHR through extracellular vesicles (sEVs) present in follicular fluid (FF) from follicles of varying sizes. In the first experiment, GCs and sEVs were collected from the FF of small (3–5 mm), medium (5.1–7 mm), and large (7.1–9 mm) ovarian follicles from Bos taurus indicus cows. In the second experiment, GCs and sEVs were collected from the FF of small (3–6 mm) and large (8–14 mm) follicles from Bos taurus taurus cows. Initially, we assessed the ability of sEVs to carry LHR mRNA by comparing its expression profiles in sEVs derived from different size follicles. Our findings revealed that as follicular development progresses, LHR levels in FF sEVs decrease, while in corresponding GCs, from which the sEVs primarily originate, show increased LHR expression. To further investigate whether GCs represent an additional source of FF sEVs carrying LHR mRNA, GC cultures were established and sEVs secreted into the culture medium (ME-sEVs) were analyzed for LHR mRNA levels. A similar pattern was observed in ME-sEVs derived from GCs of small versus large follicles, with decreased LHR mRNA levels in sEVs secreted by GCs from large follicles compared to small follicles. This suggests that LHR is likely packaged into sEVs in small follicles stage, and shuttled into follicular cells during follicular growth, preparing them for the ovulatory stimulus. Our study uncovers a possible mechanism of LHR acquisition by GCs, which involves EVs and can possibly be involved in follicle quality and ability to respond to LH stimulus.

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Abstract The aim of this study was to evaluate the probability of pregnancy in heifers weaned at different ages and bred at 13 to 15 months old. A total of 121 Braford heifers were used, weaned as calves at 77 days (early) or 147 days (conventional) of age. To develop the statistical models of reproductive performance, factors related to the development of the heifers were analyzed. The analysis included a diagnosis of multicollinearity using the Pearson correlation matrix, adjusting the model by means of the Hosmer and Lemeshow test. The response variable, rate of pregnancy, was analysed using the LOGISTIC procedure. Beginning with a weight of 271 kg and an age of 402 days at the start of the breeding season, the pregnancy rates increased by 18.4% and 29.0%, respectively for every 15 kg increase in body weight and 10-day increase in age. However, a reduction of 15 kg in body weight and of 10 days in age reduced the pregnancy rates in the heifers by 15.5% and 22.5%. An increase or reduction of 0.100 kg in the average daily gain between early weaning and conventional weaning represented an increase of 44.6% and a reduction of 30.9% in the chances of pregnancy. Early-weaned heifers require correct nutritional management to allow satisfactory postweaning weight gains so as not to compromise their reproductive performance.

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Abstract Pregnancy induces critical physiological adaptations to support embryonic development and fetal survival. This study compared endometrial and placental phenotypic and histomorphometric characteristics of Piau and Commercial sows at two gestational ages (25 and 35 days). Twelve sows (six Piau and six Commercial) were evaluated in a randomized design, with samples collected from three regions of the right uterine horn of each animal. Histomorphometric analyses were performed using microscopy and ImageJ software. Statistical analyses employed linear mixed-effects models, with Shapiro-Wilk and Levene’s tests applied to assess normality and homogeneity of variances, respectively. At 25 days of gestation, Commercial sows showed greater uterine and ovarian weights, a higher number of corpora lutea, and longer uterine horn horns, reflecting genetic selection for reproductive efficiency. Conversely, Piau sows exhibited more advanced embryonic development at this stage, with fetuses of greater size. At 35 days, the phenotypic superiority of Commercial sows persisted, while Piau fetuses maintained greater weight and length, indicating distinct temporal growth dynamics. Histomorphometric analyses at 25 days revealed that Commercial sows had increased placental connective tissue deposition and thicker endometrial epithelium, whereas Piau sows presented larger placental vascular area, as well as enhanced endometrial vascularization and glandular density across all uterine regions. At 35 days, no significant differences were observed in placental vascular area and endometrial vascularization; however, subtle trends in connective tissue development suggested ongoing placental differentiation. These findings highlight distinct reproductive strategies between Piau and Commercial sows, with potential implications for embryonic development and gestational success. Altogether, the results confirm that genetic background influences uterine and placental morphology during early gestation.

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Abstract The presence of follicles and the corpus luteum (CL) in the ovarian surface plays a key role in determining the morphological and molecular fate of the female reproductive tract. However, the specific response of the isthmus epithelium to these ovarian structures remains poorly understood. This study hypothesizes that distinct ovarian structures differentially affect both the cellular aggregate-forming capacity of oviductal isthmus cells and the expression of ESR1, ESR2, and PGR genes. Reproductive tracts were categorized into three groups: ovaries with ≤5 mm follicles (small follicles group; SF); ovaries with follicles between 8-10 mm (large follicles group; LF); and ovaries containing active corpus luteum (CL Group). Isthmus cells from the three groups were cultivated to form cellular aggregates (oviductal explants) during 24 h. Moreover, the expression levels of ESR1, ESR2, and PGR genes were analyzed in the isthmus cells of the experimental groups. The isthmus cells of LF group showed an increased number of cellular aggregates than SF and CL group. Additionally, the SF group presented more aggregates than the CL group. Gene expression analysis revealed that ESR1 expression was higher in the SF group than in the LF group. Moreover, PGR expression was greater in the CL than in the SF group, as well as in the LF than in the SF group. In conclusion, ovarian structures impact the cellular aggregate formation capacity and the gene expression of ovarian steroid receptors in isthmus cells.

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Abstract Since temperature plays an important role in feed consumption, chemical reactions and metabolism, it was hypothesized that it is below ideal it could interfere in the masculinization of tilapia in biofloc system (BFT) and the ideal methyltestosterone (MT) concentration in the feed. The masculinization rates of Nile tilapia in a zero-water exchange BFT (and without clarification) at 25°C and 28 °C combined with concentrations of MT in the feed (0 (control), 10, 20, 30 and 40 mg ∙ Kg-1 of feed) were evaluated using 3 replicates per treatment (30 tanks of 50 liters, 2 larvae ∙ L-1). Larvae were fed five times a day for 28 days. The water quality and growth performance did not diverge between MT concentrations (p > 0.05). Larvae grew 2.7 times higher in 28°C than 25°C. The control treatments did not differ from each other for male proportion (mean = 66,75%) but differed from all hormonal treatments. These treatments presented masculinization rates above 98.7% and 96.9%, at temperatures of 25°C and 28°C, respectively, and did not differ from each other at the same MT concentrations. Therefore, it is feasible to use an even lower concentration (10 mg of MT ⋅ Kg-1 of feed) in a zero-water exchange BFT, regardless of these temperatures. At the lower temperature, the input of MT in the system was smaller due to smaller feed intake, however the fingerlings would take longer to reach commercial body weight. Furthermore, after 2 hours of the last hormone feeding, MT residues were not detected in any biofloc/water mixture samples.

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Abstract Intratesticular injection of chemical agents in dogs has been investigated for nearly half a century as promising methods for nonsurgical sterilization. This study was conducted to determine the effects of formalin and calcium chloride injection in mediastinum testis on semen quality, testicular width, testosterone level and histopathological changes in dogs, and to evaluate the usability and success of the intra mediastinum testis injection technique in chemical castration. Eighteen adult male dogs were divided into 3 groups including a control (0.5 ml 0.9% saline solution), formalin (5%) and CaCl2 (50%) into the mediastinum. Testicular diameter, spermatological characteristics and testosterone concentrations were determined on days 0, 22 and 44 following applications. The dogs were castrated on 45 day and the testicles were sent to the laboratory for histopathological examinations. Formalin and CaCl2 administered groups showed apparent enlargement and firmness of the testis with symptoms of discomfort and pain. One dog in the formalin group and two dogs in the CaCl2 group developed scrotal inflammation within three days, which progressed to scrotal ulceration and fistula formation. On day 44 after treatment, it observed that sperm motility and sperm concentration in the formalin and CaCl2 groups were decreased compare to control group while dead and abnormal sperm rates were increased (P<0.05). The testosterone concentration significantly increased in CaCl2 compare to control and formalin groups on day 44 (P<0.05). In histopathological evaluations, moderate degenerative changes were detected in the testicular tissue of the formalin group. The findings of this study showed that CaCl2 and formalin injections into the mediastinum testis under ultrasonography guidance are easily to perform and could be alternative to both the intratesticular injection and the surgical technique.

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Abstract How intensive the masculinization period of Nile tilapia can be in biofloc technology (BFT) based on higher stocking densities and a zero-water exchange protocol to mitigate the environmental impact has not been studied yet and needs investigation. Thus, our objective was to determine the optimal stocking density for reduction of hormonal effluent in BFT, better growth and masculinization rate of Nile tilapia larvae, and lower total variable cost. Five stocking densities (1.5, 3, 4.5, 6, and 7.5 larvae ∙ L-1) with four replicates were tested. Tilapias were fed with 60 mg of 17α-methyltestosterone (MT) ∙ Kg-1 of feed for 28 days, and with hormone-free feed until the common selling body weight of 1g. Water quality variables showed no significant differences between treatments, except for total settleable solids, pH, and final total organic carbon, that had a negative, positive and quadratic linear pattern with the increasing of stocking density, respectively. Growth performance variables such as final body weight, specific growth rate, survival and individual feed intake (and consequently hormone input) were superior at the lower stocking density. Masculinization rate (98.87%) did not differ among treatments, but total variable cost increased to produce 1g fingerlings in higher stocking densities. Hormone residue was not detected in BFT water of any treatment 12 hours after the last feeding so, in conclusion, even being the highest hormone input in the system, 1.5 larvae ∙ L-1 is the best level of stocking density studied for tilapia masculinization in a zero-water exchange BFT.

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Abstract The objective of this study was to evaluate the effect of eCG on the time of ovulation, preovulatory follicle diameter and pregnancy rate in Holstein cows with a prolonged proestrus protocol in the Ecuadorian Amazon. This research was conducted between October 2018 to October 2019. We used 447 multiparous cows, milked once a day, with an average milk production of 8 to 9 liters cow/day. Two treatments were used, T1 with the addition of eCG (206 cows); and T2 without the application of eCG (241 cows). The pregnancy rate was higher in cows that received eCG (59.2%, T1) than in cows that did not receive eCG (45.6% T2: P<0.05), but the presence or absence of estrus did not differ (P>0.05) among groups (T1: 70.4%), T2: 65.6%). The diameter of the dominant follicle at P4 device removal (T1: 9.5 ± 0.6 mm vs T2: 9.7 ± 0.7 mm), as well as at the time of artificial insemination (T1: 11.6 ± 0.7mm vs T2: 11.2 ± 1.1mm) was not different among groups (P>0.05). However, the diameter of the dominant preovulatory follicle was greater (P<0.05) in cows in T1 (13.5 ± 1.1 mm) than those in T2 (12.5 ± 1.6 mm), with a follicular growth rate of 1.3 ± 0.1 mm/day and 0.9 ± 0.1 mm/day, for T1, T2 respectively (P<0.05). The mean ovulation time from device removal was shorter for T1 (94.7 h; P<0.05) than for T2 (101.5 h). The diameter of the CL was greater (P<0.05) for T1 (20.8 ± 2.3 mm) than T2 (19.1 ± 3.6 mm), while plasma concentrations of P4 did not differ among groups (P>0.05). In conclusion, the addition of eCG to the J-Synch protocol increased the diameter of the preovulatory follicle and the resulting CL, which favored the pregnancy rate in cows in the Ecuadorian Amazon.

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Abstract This study aimed to establish in vitro culture conditions for inducing ovulation in lambari (Astyanax altiparanae). All fish received a priming dose of either 0.6 mg/kg or 100 IU hCG/kg (human chorionic gonadotropin) in vivo. In experiment 1, 0.6 mg and 5.4 mg of carp pituitary extract (CPE)/ kg of ovarian fragment were tested as resolving doses. In experiment 2, prostaglandin F2α (PGF2α) analogue at 100 ng/mL was added to 5.4 mg CPE/kg as the resolving dose. Since ovulation did not occur, in experiment 3 and 4, we compared diverse forms of obtaining follicles, comparing manual follicle dissociation and collagenase at 100, 200, and 400 CDU/mL. Additionally, in experiment 4, the resolving dose of CPE and hCG was replaced by 1 µg/mL 17α,20β-dihydroxy-4-pregnen-3-one (DHP). Ovulation was successful only in Experiment 4, using mechanically dissociated follicles with a priming dose of either CPE or hCG and 1 µg/mL DHP as the resolving dose. Key findings include that 5% CO2 is unnecessary, mechanical dissociation of ovarian fragments is optimal, a priming dose of CPE or hCG is required, and DHP at 1 µg/mL is effective. These results establish a standardized protocol for in vitro ovulation induction in A. altiparanae, offering a valuable tool to study ovarian function and spawning failure in tropical species.

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Abstract Dichlorvos (DDVP) is a widely used organophosphate insecticide. The reports of its toxicity and induction of oxidative stress have warranted the search for an antidote. Melatonin (MLT), a hormone found naturally in the body is known to mitigate oxidative stress. Therefore, the present study aimed to investigate the protective effects of melatonin on dichlorvos-induced toxicity in the testes and epididymis. Sixty (60) 10 weeks of male Wistar rats (160 ± 10g) randomly grouped into four (n=15, A-D) were used and treated as follows: A (Control): corn oil (1 mL/kg body weight), B: MLT (10 mg/kg), C: DDVP (1.6 mg/kg), and D: DDVP and MLT. Except for the MLT intra-peritoneal treatment, other test samples were administered orally for 45 days. Histomorphometry and histological examinations were conducted on the testes and epididymis 24h, 14 days, and 45 days after treatment. There was a significant decrease in the epithelial length of the epididymis of rats treated with DDVP at 24h and 45 days post-treatment compared to the control. MLT significantly increase the mean epididymal epithelial length at 24h and 45 days post-treatment in the co-treated group. The DDVP-induced significant reduction in the testes’ luminal diameter was reversed by MLT in the co-treated group. MLT, DDVP (45th-day treatment), and DDVP+MLT (14th and 45th-day treatment) increased the epididymal luminal diameters significantly compared to the control. Meanwhile, DDVP altered both the testicular and epithelial architectures and caused atrophy of the seminiferous tubules and spermatogenic arrest with atrophy of epithelial tubules, especially following 14- and 45-day treatment. These lesions were reversed in the co-exposed rats by MLT. In conclusion, melatonin mitigates the derangements induced by dichlorvos in the testes and epididymis.

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Abstract The endocrine regulation of testicular and ovarian development during early life in chelonians remains poorly understood, despite major morphophysiological changes occurring in this period. This study investigated the immunolocalization of estrogen (ER1 and ER2), androgen (AR), and aromatase (AROM) in the gonads and mesonephros of Podocnemis expansa during the first year after hatching (n = 5; males: 1, 3, 12 months; females: 2, 4 months). After euthanasia, gonads and mesonephros were collected, fixed in 10% buffered formaldehyde, and processed for immunohistochemistry. The intensity of immunoreactions varied according to age and sex, but not with tissue distribution. AROM, AR, ER1, and ER2 were detected in both gonads and mesonephros, confirming the persistence of steroidogenic and steroid-responsive activity after hatching. In males, AROM showed weak to moderate labeling in germ and interstitial cells of seminiferous tubules, whereas ER2 expression predominated in spermatogonia and interstitial endocrine cells. In females, AROM and AR were moderately expressed in the germinal epithelium and oogonia at two months, decreasing at four months, while ER2 persisted in follicles and oogonia. These findings suggest a dynamic endocrine environment influencing posthatch gonadal maturation, with the mesonephros acting as a transient extragonadal source of steroids. Despite the small, sex-unbalanced sample (due to lack of dimorphism), the results provide valuable baseline information and support future quantitative and functional studies on the reproductive endocrinology of P. expansa and other chelonians with temperature-dependent sex determination.

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Abstract The yolk sac (YS) is a multifunctional and essential structure for early maternal-fetal interactions, playing roles in nutrient supply, organogenesis, immune modulation, and hematopoiesis. Unlike most species, dogs maintain the YS until the end of pregnancy, yet little is known about its functional activity, particularly in later stages. Animal models are crucial for exploring maternal-fetal processes and offering insights into comparative development, including in humans. This study aimed to analyze the YS structure in canine embryos and fetuses at early (up to 20 days, n = 6), middle (21–40 days, n = 6), and late (41–60 days, n = 6) stages. Gestational age was estimated using crown-rump length (CRL) and external anatomy. Samples from the university’s Veterinary Hospital and neutering campaigns in Londrina (Paraná, Brazil) were fixed in 10% formaldehyde for morphological analysis (H&E, PAS, and Picrosirius red) or 4% paraformaldehyde for OCT4 immunostaining (CEUA/UEL protocol number 029.2022). Histologically, the YS showed a trilaminar structure with blood islands in the intermediate layer surrounded by mesenchyme. Within these islands, undifferentiated cellular niches (OCT4+) were identified. During gestation, the YS changed from a thin, smooth, pale-red membrane to a wrinkled structure with narrowed ends and a thickened central region, resembling a "whale tail." The red color, indicative of vascularization, intensified with blood island enlargement. Collagen secretion, particularly type I, progressively increased around the blood islands. Glycoprotein deposits persisted until late gestation, as shown by PAS staining. These findings highlight the structural, secretory, and reserve roles of the canine YS throughout pregnancy.

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Abstract Surgical techniques in aquatic species are underdeveloped, despite these species comprising a significant superclass among vertebrates. In the context of species preservation, studies involving the use of reproductive tissues typically involve the euthanasia of donors. Thus, in animals at high risk of extinction, the practice of sacrificing them presents a point of contradiction between ex situ conservation efforts. The objective of this study was to compare the viability of spermatogonia stem cells (SSCs) collected from Brycon orbignyanus using conventional methods (euthanasia) versus a surgical procedure. Lateral celiotomy was performed on 27 immatures males to obtain a portion of gonadal tissue. The fish were divided into three groups (n=9), with each group receiving polyglactin 910, polyester, or catgut sutures, respectively, for celiorrhaphy. Dermorraphy was performed using nylon sutures in all groups. A. An additional euthanasia group consisted of nine animals exposed to 20 mg/L of propofol. The survival rate over a 48-hour period was 100% for the polyglactin 910 group and 77.7% for the polyester and catgut groups. Additionally, the viability of SSCs was similar between the euthanasia and surgical procedures. The lateral celiotomy technique is feasible for obtaining SSCs in fish with laterally compressed anatomy, such as B. orbignyanus. Additionally, the technique allows the preservation of SSCs as a model for endangered fish species.

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Abstract Serum calcium fluctuations are common during the peripartum period of dairy cattle and several studies have attempted to demonstrate the impact of decreased blood calcium (Ca) on subclinical endometritis; however, the highly dynamic and complex nature of the peripartum period in dairy cows may impair the establishment of the cause-and-effect relationship. The objective of this review is to compile information regarding hypocalcemia and subclinical endometritis and their relationship, as well as the available in vivo and in vitro study models that artificially induce subclinical states of hypocalcemia and endometritis in cows that are not in peripartum period. Regarding hypocalcemia, several studies have demonstrated the effectiveness and safety of protocols using Ca chelators such as ethylenediaminetetraacetic acid (EDTA) or ethylene glycol tetraacetic acid (EGTA) in vivo. The induced transitory hypocalcemia impaired feed intake, rumination and neutrophilic phagocytic and oxidative burst response. However, the effects on uterine environment remain poorly explored. Although these experimental models allow the understanding of the effects of hypocalcemia alone, without the peripartum metabolic and hormonal variations, the effects are likely underestimated because dairy cows may experience hypocalcemia for much longer periods. For studying bovine endometritis, the main experimental in vivo model is the intrauterine infusion of pathogenic bacteria or their components (lipopolysaccharide - LPS), which induce endometrial inflammation, even causing long-term negative effects. Several in vitro and ex vivo models have also been developed, which are mainly indicated to investigate the mechanisms underlying endometrial inflammation in cattle because there is no interaction with other tissues, organs and systems, as would occur in vivo. In conclusion, current models still face limitations and, therefore, future efforts to the development and refinement of in vivo and in vitro experimental models are necessary.