Eggshell translucency: its relationship with specific gravity and eggshell color and its influence on broiler egg weight loss, hatchability, and embryonic mortalities.

Eggshell quality is among the most important factors affecting hatchability in broiler breeders, and therefore several methods for its assessment are available in the poultry industry. Among them, eggshell translucency has received special attention in recent years due to its connection with ultrastructural disorganization of the shell layers. However, there is very limited data on the impact of translucency on hatching eggs and on the possible links between this trait and specific gravity (SG) or shell color. Thus, our study investigated associations and interactions between eggshell translucency, SG, and color on incubation parameters of eggs from the same breeding flock (Ross 308AP, 51 wk of age). To this end, light and dark eggs within 5 different SG categories (≥1.065, 1.070, 1.075, 1.080, and ≤1.085) were selected from 15,976 eggs, graded into 3 translucency scores, and later incubated to evaluate egg weight loss, hatchability and embryonic mortalities. In general, translucency scores were evenly distributed within SG categories (χ2 [8, N = 1,138] = 13.67, P = 0.090) and color (χ2 [2, N = 1,138] = 4.93, P = 0.084). No interactions between eggshell translucency and SG or between translucency and color were found for the analyzed variables. An interaction was observed between SG and eggshell color for the variable egg weight loss, where the light-shelled eggs, in most SG categories lost more weight throughout incubation than dark eggs. Eggshell translucency affected egg weight loss, hatchability, and embryonic mortality on 11 to 18 d of incubation, with highly translucent eggs showing the worst results. At the same time, eggs with SG lower than 1.070 displayed the greatest weight loss, lowest hatchability, and highest contamination. We found no influence of eggshell color on weight loss or hatchability, but light-shelled eggs exhibited higher late embryonic mortality. Together, these data suggest that despite its effects on certain hatching parameters, shell translucency bears no relationship to SG or color.

Successful xenotransplantation of testicular cells following fractionated chemotherapy of recipient birds.

An essential step in the success of germ cell transplantation is the preparation of the recipient's testicular environment to increase the availability of stem cell niches. However, most methods for this purpose in birds face serious limitations such as partial germ cell depletion, high toxicity and mortality, or the need to use expensive technologies. Here, we validated a simple and practical technique of transferring quail testicular cells into chicken testes depleted of endogenous spermatozoa by fractioned chemotherapy (20 mg/kg/week busulfan for 5 weeks). This protocol resulted in a very low mortality of the treated day-old chicks and, despite maintenance of androgenic activity, sperm production was decreased by 84.3% at 25 weeks of age. NANOG immunostaining revealed that very few to no germ cells were present following treatment with 20 and 40 mg/kg, respectively. RT-qPCR data also showed that c-MYC and NANOG expression declined in these treatments, but GRFα1 and BID expressions remained unaltered among groups. After xenotransplantation, quail germ cells were immunodetected in chicken testes using a species-specific antibody (QCPN), and quail ovalbumin DNA was found in seminal samples collected from chicken recipients. Together, these data confirm that fractionated administration of busulfan in hatchlings is a practical, effective, and safe protocol to prepare recipient male birds capable of supporting xenogeneic spermatogenesis.

Ecological drivers of breeding periodicity in four forest neotropical eagles.

We explore the potential factors that affect clutch initiation in four Neotropical large raptors (Harpy eagle-HE, Crested eagle-CE, Ornate hawk-eagle-OHE, and Black hawk-eagle-BHE) by analyzing 414 clutch events mostly obtained from captive individuals. Differences in how clutch initiation is associated with changes in photoperiod were found between HE and both hawk-eagles, and between CE and BHE. Changes in temperature at the time of clutch initiation only differed between HE and OHE, whereas changes in precipitation varied between BHE and all other species. Principal Component Analysis of these environmental cues showed that ellipses in the dataset of each species overlap, but only ellipses from CE and OHE had the same variation trends. This means that although these species live under similar ecological conditions, they exhibit three different patterns of response to environmental cues. Apparently, these patterns are not associated with phylogenetic relatedness because species belonging to the same clade do not show the same response pattern. Diet diversity analysis revealed that HE has the least varied diet, and CE and OHE the most varied diet. The fact that species who fit the same reproductive timing response to environmental cues show similar diets leads us to hypothesize that breeding in these eagles was most likely shaped by food availability.

Comparative fecal steroid profile during pregnancy, parturition, and lactation between natural fertilization and embryo transfer in ocelots (Leopardus pardalis).

Despite the invaluable role that assisted reproduction technologies (ARTs) play in conservation, pregnancy and parturition rates by embryo transfer (ET) are low for most endangered felids. Thus, efforts to expand the knowledge on pregnancy biology and ET are still required. In this context, we examined fecal sex steroid metabolites (i.e., estrogens, glucocorticoids, and progestogens) of eight ocelots submitted to natural fertilization (NF) and ET in 22 pregnancies (19 NF and 3 ET). Fecal samples were collected and assessed for each pregnancy from estrous cycle, pregnancy, and lactation, totaling 155 days. In short, progestogen levels remained high and unchanged (P < 0.05) from conception until parturition for females maintained under NF. On the other hand, females submitted to ET exhibited changes (P > 0.05) in progestogen levels from conception until parturition, with a significant decrease during pregnancy (480.72 ng/day; r2 = 0.81; P < 0.0001). Significant changes between NF and ET also were noted in estrogen levels between the first and last thirds of pregnancy (P < 0.05), in which estrogen levels exhibited a negative correlation (P < 0.01) between themselves. Regarding glucocorticoids, significant changes (P < 0.01) were observed only in the first third of pregnancy between NF and ET, which we believe may be related to the handling for ovarian synchronization and ET. Besides hormonal changes, the pregnancy was more prolonged (2.5 days) and more prone to dystocia in ET than NF. Overall, 24 embryos were transferred into eight females (3/1), with three kittens being born from three distinct deliveries (i.e., 12.5% of embryos and 37.5% of females). Our findings have supported the great potential of production and transfer of long-term frozen embryos in ocelot conservation. However, they reveal possible effects of these biotechnologies on hormonal levels during pregnancy linked with low conception and parturition rates and dystocic cases in felids.

Beneficial influence of fetal bovine serum on in vitro cryosurvival of chicken spermatozoa.

Chicken spermatozoa are highly susceptible to cryopreservation often requiring extenders containing additives to enhance their post-thaw quality. Although protective properties of fetal bovine serum (FBS) during freezing of tissue cultured cells are widely known, its potential as a cryoprotectant for sperm cells has not been largely explored. Thus, the aims of our study were to (i) investigate the protective effect of FBS at different concentrations (0, 5, 10, 15 and 20%) against cryodamages in chicken spermatozoa, and (ii) test the FBS concentration that yielded the best preservation versus 1 mg/mL of cholesterol-loaded cyclodextrins (CLCs). Samples were assessed before and after freezing for sperm motility parameters, plasma membrane and acrosomal integrities, mitochondrial membrane potential, oxidative stress and plasma membrane peroxidation. Our findings showed that, despite their beneficial effects on fresh spermatozoa, higher FBS concentrations (15 and 20%) obtained the worst results for most motility and functional parameters after thawing. In contrast, lower FBS concentrations (5 and 10%) improved all post-thaw variables when compared to control. Afterwards, based on regression analysis, the concentration of 7% FBS was chosen to be assessed against CLCs in an experiment composed by four groups: control, FBS, CLCs, and FBS + CLCs. FBS and FBS + CLCs groups exhibited higher progressive motility in fresh samples, whereas only FBS maintained higher post-thaw progressive motility. Additionally, the incorporation FBS into extenders increased the percentage of rapid cells and reduced free radicals production and plasma membrane peroxidation. Together, these outcomes indicated that FBS minimize some harmful effects of cryopreservation, providing an alternative for chicken semen extenders that in many aspects appears to be superior to CLCs at 1 mg/mL.

Cryopreservation of margay (Leopardus wiedii) spermatozoa: Effects of different extenders and frozen protocols.

Sperm cryopreservation offers many benefits to wild felids conservation programs. However, the implementation of these programs is limited by the different responses of each species to the cryopreservation protocols and extenders used, requiring the formulation of species-specific protocols. For this purpose, semen samples from 6 margays (Leopardus wiedii) were submitted to 2 cryopreservation protocols: 1) manual freezing (cooling rate of - 0.33 °C/min at 5 °C/180 min and freezing rate with two steps - 9 °C/min for 2 min and -19.1 °C/min for 2 min) and 2) automatic freezing machine (cooling rate of - 0.25 °C/min at 5 °C/120 min and freezing rate with one step -20 °C/min for 8.3 min) using 2 commercial extenders, an egg yolk-based (Test Yolk Buffer; TYB) and an egg yolk-free extender (AndroMed; MED). Post-thawed sperm quality was assessed at 3 time points (immediately after thawing and 1 and 2 h post-thawed) by sperm motility index (SMI), plasma membrane and acrosomal integrity, and mitochondrial membrane potential (MMP). Regarding SMI, TYB yielded superior results (29.4 ± 3.5%) compared to MED (11.2 ± 2.8%; p < 0.002) immediately after thawing until 2 h after thawing (TYB 3.9 ± 1.7% and MED 0.0 ± 0.0%; p < 0.05). Furthermore, the automated freezing method provided higher motility compared to the manual freezing procedure immediately post-thaw (25.08 ± 3.66% and 15.78 ± 3.29%, respectively) and 1 h post-thaw (13.71 ± 2.56% and 6.03 ± 1.97%, respectively; p < 0.05). The percentage of intact acrosomes and plasma membranes and the percentage of sperm with high MMP were superior for TYB when compared to MED regardless of cryopreservation protocol (p < 0.05). Conversely, the interaction between cryopreservation protocols and extenders was observed for MMP where TYB exhibits better results compared to MED (p < 0.05) in both procedures, but it was higher in automated procedures. For MED, no changes were found in MMP between procedures. Considering only TYB, samples showed higher MMP when submitted to an automated procedure (p < 0.05). In conclusion, the slow cooling rates with shorter time of exposure to glycerol contributed to minimize cryodamage in the Margays' sperm. Moreover, results indicated that association between TYB and automatic freezing machine ensured the minimal quality of spermatozoa after thawing required for further use in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI).

Assessment of different sperm functional tests in golden-headed lion tamarins (Leontopithecus chrysomelas).

The golden-headed lion tamarin (Leontopithecus chrysomelas) is an endangered species endemic to Brazil's Atlantic Forest, a shrinking biodiversity hotspot. As in other Neotropical primates, its semen characteristics and freezability are poorly studied. Hence, reproductive technologies for callitrichids would greatly benefit from reliable methods of semen analysis. In a bid to promote reproductive research in tamarins, we validated simple and inexpensive sperm function tests that can be used to monitor sperm-egg binding, plasma membrane and acrosome integrity, mitochondrial activity, and DNA fragmentation. Ejaculates from adult males were individually diluted and divided into control and damage-induced aliquots, and then samples comprising assorted amounts of damaged spermatozoa were examined by organelle-specific tests. Our findings showed that sperm-binding in chicken egg perivitelline membrane (EPM) positively correlated with the number of spermatozoa injured by snap-freezing. Eosin-nigrosin (EN) and propidium iodide readings were correlated with each other, and both provided robust measurements of plasma membrane integrity. A high correlation between expected and measured amounts of acrosome-intact spermatozoa was found using Fast Green-Rose Bengal (FG-RB), Coomassie Blue (CB), and FITC-PSA stains, and all three methods exhibited comparable results. Likewise, different percentages of UV-irradiated spermatozoa were accurately assessed for DNA integrity by Toluidine Blue (TB) and sperm chromatin dispersion (SCD) tests. Comparisons between 3,3'-diaminobenzidine (DAB) and JC-1 stains also indicated the reliability of the former assay to ascertain gradual increases in spermatozoa with greater mitochondrial function. These data confirmed that different parts of the tamarin spermatozoa can be simply and consistently evaluated by EPM, EN, FG-RB, CB, TB, and DAB protocols.

Contenção farmacológica da jaguatirica, Leopardus pardalis, para coleta de sêmen, pela associação de tiletamina-zolazepam e xilazina / Chemical restraint of ocelot, Leopardus Pardalis, for semen collection with the association of tiletamine-zolazepam and xylazine

Foram avaliados os efeitos anestésicos da associação de cloridrato de tiletamina (7 mg/kg), cloridrato de zolazepam (7 mg/kg) e cloridrato de xilazina (1 mg/kg), doses calculadas para peso estimado, aplicados por via intramuscular profunda para a contenção farmacológica de machos de jaguatirica, (Leopardus pardalis. Linnaeus, 1758) (Felidae), submetidos à coleta de sêmen por eletroejaculação. Quatro animais foram anestesiados em quatro a cinco ocasiões diferentes, com intervalo mínimo de 30 dias, totalizando 17 procedimentos. Em algumas ocasiões, quando a contenção farmacológica inicial não propiciava segurança para a manipulação do animal, foi necessária a aplicação complementar de anestésicos, sendo então administrada 1/3 da dose inicial, fato necessário em apenas três (17,64%) do total de procedimentos. Após a administração das drogas, foram monitorados durante 80 minutos parâmetros como frequência cardíaca, frequência respiratória, temperatura retal, miorrelaxamento e nocicepção. O período de latência foi de 3,56±0,55 minutos após a injeção e o tempo para recuperação foi de 169,60±12,37 minutos. Não foi evidenciada diferença estatística (p>0,05) entre os animais e os momentos avaliados para reações dolorosas, miorrelaxamento, bem como para os parâmetros: frequências respiratória e cardíaca e temperatura retal. A associação de tiletamina-zolazepam e xilazina, nas doses de 7mg/kg e 1mg/kg respectivamente, proporcionou uma boa qualidade anestésica, curto período de latência, bom período anestésico hábil e com segurança para a realização do procedimento de eletroejaculação nas jaguatiricas, com boa qualidade do sêmen obtido.

The anesthetic effects of tiletamine hydrochloride (7 mg/kg), zolazepam hydrochloride (7 mg/kg) and xylazine (1 mg/kg) association, doses calculated by the estimated weight, were evaluated after deep intramuscular injection for pharmacological restraint of male ocelot (Leopardus pardalis. Linnaeus, 1758) (Felidae), subjected to semen collection by electroejaculation. Four animals were anesthetized on four to five different occasions with an interval of 30 days, totaling 17 procedures. In some instances, when the initial pharmacological restraint was not propitiated safety for handling the animal, it was necessary to apply supplementary anesthetics, and then administered 1/3 of the initial dose, fact necessary only in three procedures (17.64%). After the injection, parameters like heart rate, respiratory rate, rectal temperature, muscle relaxation and nociception was monitored for 80 minutes. The latency period was 3.56±0.55 minutes after injection and the time for recovery was 169.60±12.37 minutes. There was no significant statistical difference (p>0.05) between the animals and moments evaluated for painful reactions, muscle relaxant, as well as for the parameters: respiratory and heart rates and rectal temperature. The combination of tiletamine-zolazepam and xylazine at doses of 1mg/kg and 7mg/kg respectively, provided a good quality of anesthesia, short latency period, good viable anesthetic period and safety to proceed the electroejaculation in ocelots, with good quality of obtained semen.