Animal Reproduction Science最新文献

As sperm cryopreservation and other assisted reproductive technologies (ARTs) advance in common amphibian species, focus on applying non-lethal sperm collection methods to the conservation and genetic management of threatened species is imperative. The goal of this study was to examine the application of logistically practical ART protocols in a threatened frog (Litoria aurea). First, we tested the efficacy of various concentrations of human chorionic gonadotropin (hCG) (20, 40 IU/g bodyweight) and Gonadotropin releasing hormone antagonist (0.25 µg/g and 0.5 µg/g body weight GnRH-a) on the induction of spermatozoa. Using the samples obtained from the previous trials, we tested the effect of cold storage and cryopreservation protocols on long-term refrigerated storage and post-thaw sperm recovery. Our major findings include: (1) high quality sperm were induced with 20 and 40 IU/g bodyweight of (hCG); (2) proportions of live, motile sperm post-thaw, were recovered at higher levels than previously reported for L. aurea (>50%) when preserved with 15% v/v DMSO and 1% w/v sucrose; and (3) spermic urine stored at 5 °C retained motility for up to 14 days. Our findings demonstrate that the protocols developed in this study allowed for successful induction and recovery of high-quality spermatozoa from a threatened Australian anuran, L. aurea, providing a prime example of how ARTs can contribute to the conservation of rare and threatened species.

Sperm adhering to glass slides is one of the main problems during fish sperm motility analyses with CASA systems. To mitigate this, albumin is the supplement added most frequently to activating solutions. However, there is no data on the use of supplements other than albumin (in various concentrations) in analyses of European whitefish (Coregonus lavaretus) sperm motility. This issue was investigated in the presented research using three anti-adhesive supplements (albumin, casein, Pluronic F-127) that were added to Billard solution (BS: 20 mM Tris, 1 mM CaCl₂, 154 mM NaCl, 30 mM glycine at pH 9.0) at different concentrations (0.0; 0.1; 0.2; 0.5; 1.0; 2.0%). It was noted that the addition of the lowest concentration (0.1%) of albumin, casein, or the pluronic to BS had a significant effect on the motility and kinetic parameters of whitefish sperm compared to pure BS. BS supplemented with 0.2–0.5% albumin was the most appropriate variant used for whitefish sperm motility activation in the present experiment. BS supplemented with the pluronic at 1.0–2.0% concentrations resulted in significantly higher values of almost all CASA parameters compared to casein at the same concentrations. Moreover, CASA parameters determined in this variant of the pluronic (1.0–2.0%) were similar to those when BS was supplemented with the same albumin concentrations. This indicated that instead of albumin, the pluronic at higher concentrations in BS might be used to analyze whitefish sperm motility.

The aim of the present study is to evaluate the effect of Moringa oleifera leave extract (MOLE) on buffalo bull cryopreserved semen quality and fertility. Sixty ejaculates were collected from ten fertile buffalo bulls on a weekly basis for 6 weeks (n = 10 bulls & n = 60) then semen samples were pooled and divided into five groups. The semen of the control group was without additives. The semen of other groups was supplemented with MOLE at doses of 200, 400, 600 and 800 µg/ mL, respectively. One hundred thirty multiparous buffaloes were artificially inseminated with semen supplemented without or with MOLE at dose of 600 µg/ mL. Inclusion of MOLE in semen extender at dose 600 µg/ mL significantly elevated the total motility, progressive motility, membrane integrity and fertilization capacity of the post-thawed spermatozoa, as well as the total antioxidant capacity. However, it significantly decreased acrosomal defects of spermatozoa, and the concentration of malondialdehyde. This study indicated that inclusion of MOLE to semen extender improved the quality and fertility of the post-thawed buffalo bulls’ semen through enhancing the activities of the antioxidant enzyme system and decreasing cryodamage of the buffalo bull spermatozoa.

Studies in cows have reported that ovulation, steroidogenesis and angiogenesis are affected by stress and consequently fertility decreases. The purpose of this study was to evaluate the effects of ACTH administration during the preovulatory period on the expression of growth factors (CD-31, PDGF-A, PDGF-B, VEGFA-164, VEGFA-164b, VEGF-R1 and VEGF-R2) associated with the angiogenic process by immunohistochemistry in cows (n = 14). Results evidenced the expression of these growth factors in theca and granulosa cells from antral, atretic and dominant preovulatory follicles of ACTH-treated cows, suggesting that, under stress conditions, their expression continues to be required. VEGFA-164, VEGF-R1 and VEGF-R2 expression was greater in theca cells of dominant preovulatory follicles of the ACTH-treated group than in those of the control group. CD-31 protein expression was lower in the dominant preovulatory follicles of the ACTH-treated group than in those of the control group. PDGF-A and PDGF-B expression did not differ between groups, either in granulosa or in theca cells. These results suggest that VEGFA-164, its receptors and CD-31 are actors in the normal cycle of the ovaries and could have greater pathophysiological importance in the altered angiogenic process and other events that occur during anovulation and stress conditions. This dysregulation reinforces the importance of the angiogenic process in the pathophysiology of cystic ovarian disease in cows. This is the first report on the expression and localization of components of the VEGF and PDGF systems and CD-31 in cells from dominant preovulatory follicles after ACTH administration.

Increases in temperature can affect the reproduction of fish by decreasing the quality of gametes for fertilization. Therefore, this study aimed to evaluate the in vivo effect of temperature on the production and sperm quality of Colossoma macropomum, which is an economically relevant species for Brazil, and other countries. Broodstock were exposed for 10 days at temperatures of 29 °C (n = 4) and 35 °C (n = 4). After exposure, semen was collected and sperm quality was evaluated for kinetic, biochemical, morphological parameters, membrane integrity, and oxygen consumption. The sperm quality of males of specimens of C. macropomum exposed in vivo to the higher temperature was compromised and showed a difference in all the analyses (P < 0.05). Sperm showed decreased motility (51.9 ± 11.6 s) compared to the control (61.3 ± 5.5 s); curvilinear (CLV), straight line (SLV), and average path (APV) velocities decreased, and straightness (STR) and beat cross frequency (BCF) increased; oxygen consumption decreased (32.8 ± 3.4 pmol. (s.ml)⁻¹, picomol per second per ml) compared to the control (46.6 ± 3.5 pmol. (s.ml)⁻¹); and the activity of catalase (CAT) and glutathione S-transferase (GST) enzymes increased and decreased, respectively, in sperm from fish exposed to high temperatures. Furthermore, an increase in non-viable sperm with damaged membranes and sperm with altered morphology was observed. The observed alterations indicate that C. macropomum confined in environments with high temperatures possibly produces more sperm that are unviable for the fertilization process.

Transport-related vibrations (TV) compromise the quality of conventionally stored (17 °C) boar semen, but knowledge about TV effects after 5 °C transport is insufficient. This study evaluates the effects of TV after novel 5 °C transport compared to a 17 °C control. Ejaculates of 18 fertile Piétrain boars, diluted in a split sample procedure using Androstar Premium® (AP, 5 °C storage) or Beltsville Thawing Solution (BTS, 17 °C storage), were subjected to transport simulation using a laboratory shaker IKA MTS 4. The timing was set according to the respective processing protocols: for 17 °C BTS samples, TV simulation was performed the day of collection, 5 °C AP samples were subjected to TV the day after collection following completion of the established cooling curve to 5 °C. Six samples per ejaculate were exposed to different TV durations (0 h, 3 h, or 6 h) to evaluate the effect on sperm quality (progressive motility (PM), thermo-resistance test (30 and 300 min incubation at 38 °C (TRT30/TRT300)), mitochondrial activity (MITO), plasma membrane and acrosome integrity (PMAI)). Generalized linear mixed models revealed TV (P = 0.021) and storage time (P < 0.001) dependent declines in PM. Direct, pairwise comparisons revealed that 5 °C samples are not affected by TV (P(3 h vs. 6 h transport) = 1.0; P(0 h vs. 6 h transport) = 1.0). They therefore showed superior quality maintenance after TV compared to 17 °C samples (P(3 h vs. 6 h transport) = 0.025; P(0 h vs. 6 h transport) < 0.001). Concluding, low-temperature transport is possible without significant semen quality loss and with better quality maintenance than standard transport.

The common tench is an important thermophilic freshwater fish used for aquaculture diversification. This species is farmed in ponds as well as caught in the wild in many European and China. Artificial reproduction under controlled conditions is one of the most important part of modern aquaculture. In this study, two experiments were conducted in which the influence of hormonal agents (Ovopel and Ovaprim - experiment I) and the origin of the spawning stock (wild and pond-cultured - experiment II) on the success of reproduction in terms of ovulation rate (%), latency time (h), pseudo-gonadosomatic index (PGSI, %), hatching rate (%), abnormal larvae rate (%), and spawning efficiency coefficient (Se) were examined. While the wild fish could not reproduce in the absence of hormonal treatment with 0 % ovulation rate. In all tested hormonal treatment allowed to obtain high-quality eggs with hatching rates of over 83 %. Significant differences in latency time were observed among groups from different origins, such as differences in ovulation rate (79 % vs. 90 %, for pond-cultured and wild fish, respectively), PGSI (8.2 vs. 8.9), and hatching rate (63.8 % vs. 84.3 %), which showed significantly better quality in fish from the wild than those from pond culture. This means that when spawners of common tench of various origins are available, it is better to choose spawners from natural waters for artificial reproduction.

The biological effects of simulated photoperiod and melatonin on the control of reproduction of guinea fowls (Numida meleagris) are not well understood. Herein, thirty (30) sexually mature guinea fowl cocks were randomly assigned to 1–6 groups (n = 5) and subjected to different photoperiodic regimes in the presence or absence of exogenous melatonin (Mel; 1 BW/day, i/m) for eight weeks. Testes of the euthanized cocks were processed for gross morphology, histological, histochemical, and oxidative stress markers. Testosterone concentration was determined in serum samples using the enzyme-linked immunosorbent assay (ELISA) technique. We observed an increase in testicular size in the Mel and Non-Mel groups under long-day (LD) photoperiods, and in the Non-Mel group under short-day (SD) photoperiod. Conversely, the testicular size was drastically reduced in the Mel group for SD. Seminiferous tubules in the Mel and Non-Mel groups of the SD showed cytomorphological changes, including degenerated cells, focal vacuolations, and depletion of germinal epithelium. However, the germinal epithelium appeared to be complete and active in both the Mel and Non-Mel groups for the LD. In all groups, the testes showed positive staining for PAS with varying intensities. There was a significant difference in PAS-staining intensity between different photoperiodic regimes and exogenous melatonin. The study observed the interaction between photoperiods and exogenous melatonin on glutathione reductase (GSH), malondialdehyde (MDA), and serum testosterone. Overall, the results indicated that a long-day (LD) photoperiod, combined with exogenous melatonin, enhanced reproductive activity in male guinea fowl by increasing testicular size and serum testosterone concentration.

The present study aims to establish the morphological, morphometric, and immunostaining patterns of the steroidogenic enzymes 17β-HSD and 5α-reductase and androgen receptors (AR) during the prenatal development of the male gonad and epididymis of Cavia porcellus. Fetuses at 22, 25, 30, 40, 45, 50, and 60 days of gestation (DG) were used. Specimens were dissected and subjected to macroscopic, histological, histomorphometric, and immunohistochemical analyses. Genital and scrotal protrusions were identified in 22 DG embryos. Gonocytes were identified at 25 DG and the formation of primary testicular cords was observed at 30 DG. Through anatomical evaluation, we observed differentiation of the epididymis into the head, body, and tail at 45 DG. During development, there is a progressive decrease in the diameters of the testicular cords and epididymal ducts. 17β-HSD enzyme immunostaining was observed in Leydig cells at all ages, while 5α-reductase was observed in Leydig cell cytoplasm and gonocytes at 40, 50, and 60 DG. AR shows gonocyte labeling at 30 DG. Thus, from the second trimester of pregnancy, it is possible to observe patterns of anatomical development, such as genital and scrotal prominence (22 DG), the appearance of gonocytes in the testicular cords at 25 DG, and the beginning of the organization of primary testicular cords at 30 DG, suggesting sexual differentiation. The 17β-HSD, 5α-reductase, and ARs play an essential role in sexual development and differentiation, presenting immunostaining at different reproductive process times.

Follicular atresia (FA) has been assumed to serve different functions in reptiles, e.g. helping to develop hierarchies, limiting clutch size, and regression of ovarian structures. Reproductive output is dependent on a balance between ovulations and FA. Excessive rates of FA may not only be detrimental for the survival of a population, but have also been associated with pathological conditions. In order to gain insights into the physiological and potentially pathological processes of FA, we performed a decriptive study on the morphological features of the ovaries in sexually mature female veiled chameleons (Chamaeleo calyptratus, VC). Of 60 clinically healthy female VC with continuous ovarian cycling and at least one confirmed cycle with FA over at least 1.5 years, 30 were selected for macroscopic evaluation of ovarian appearance and 7 were subjected to histology and immunohistology. While FA of previtellogenic follicles happened at a low rate, expected for a species with two germinal beds per ovary and polyautochronic reproductive pattern, atresia in the late vitellogenic stage affected entire generations of follicles, consequential to ovulatory failure. Histologically, no pathological processes were identified in any of the animals. Rather, three stages of FA (early, middle, late) were defined and vitellogenic follicles showed two distinct morphological types of FA: yolky and cystic. Yolky FA was found in 21/30 (70%) animals, while cystic FA co-occurred in 9/30 (30%) of the animals.