Effects of Saccharides Supplementation in the Extender of Cryopreserved Rooster (Gallus domesticus) Semen on the Fertility of Frozen/Thawed Spermatozoa

The aim of this study was to create balanced media for the cryopreservation of rooster semen in pellets to maintain the functional state of the sperm after thawing. Fructose was replaced by trehalose in experimental media in proportions of 10% (LCM-T10) and 20% (LCM-T20), while LCM was used as a control. After artificial insemination of the hens, the eggs were incubated (n = 400). To determine the functional safety of spermatozoa in the genital tract of hens after 5, 10, and 15 days from the last insemination, we used a method for assessing the interaction of sperm with the perivitelline membrane. Significantly higher rates of egg fertilization (82–86%) were obtained when using LCM-T10 and LCM-T20 compared to control (79%, p < 0.05). Egg fertility on the 5th day from the last insemination with the LCM-T20 diluent reached 100% versus 86% in the control; on the 10th day, the fertility rates were 55% versus 20%, respectively. The best results for fertility duration were obtained by freezing spermatozoa with LCM-T20 medium. The numbers of interaction points of spermatozoa with the perivitelline membrane were as follows: on the 5th day from the last insemination with LCM-T20—461.5 ± 11.5 holes/cm2 (LCM-control—13.7 ± 2.7 holes/cm2), p < 0.01; on the 10th day with LCM-T20—319.3 ± 12.9 holes/cm2 (LCM-control—14.9 ± 3.5 holes/cm2); and on the 15th day with LCM-T20—345.2 ± 11.1 holes/cm2 (LCM-control—0 holes/cm2). In conclusion, the use of trehalose in LCM diluent medium can increase the fertility of frozen/thawed sperm and the duration of their fertility in the genital tract of hens.

34 Cellular effects of antifreeze proteins type I and III in extender for sheep semen cryopreservation

Antifreeze proteins (AFP) have been included in extenders for sperm cryopreservation to prevent ice crystal formation. Thus, this study assessed the effects of supplementing semen extender with two concentrations of AFP types I and III on the quality of frozen–thawed ram sperm. The hypothesis is that various types and concentrations of AFP enhance cryopreservation of ram sperm. Semen was collected from 4 rams, pooled in 6 replicates, and allocated into 1 of 5 treatments: Control (CONT, without AFP); AFP type I [0.1 (AFPI-0.1) or 0.5 (AFPI-0.5) µg±mL−1]; or AFP type III [0.1 (AFPIII-0.1) or 0.5 (AFPIII-0.5) µg±mL−1]. Straws were placed on a metal wire net frame at 37°C and placed in a refrigerator for 2h to cool them to 5°C (−0.25°C/min). After 2h for stabilisation, straws were cooled in liquid nitrogen vapor (−15.3°C/min) and subsequently immersed (−196°C). After thawing, samples from each treatment were evaluated microscopically (sperm kinetics, plasma membrane integrity, capacitation, hypoosmotic test, acrosome status and mitochondrial activity, chromatin condensation, morphology, binding to egg perivitelline membrane, and lipoperoxidation quantification). The normal distribution of residuals was determined by Shapiro-Wilk test and homoscedasticity by Levene’s test. Normally distributed variables were analysed with one-way analysis of variance (ANOVA), followed by Tukey’s test. The non-normally distributed were analysed by Kruskal–Wallis and Dunn’s test. The repeated-measures ANOVA in general linear model (GLM) was used to effects of concentration for each AFP type in paired samples. The Greenhouse-Geisser test was applied when sphericity was not considered, followed by the Sidak test. Values of P&lt;0.05 were considered significant. Treatments affected (P&lt;0.05) kinetic parameters, plasma membrane integrity, and morphology. Linearity was greater in AFPI-0.1 (56.6±3.1%, mean±s.e.m.), AFPI-0.5 (56.9±2.2%), and AFPIII-0.5 (64.7±6.2%) than in CONT (36.8±3.0%). Straightness was greater in all AFP-supplemented extenders (overall mean, 78.6±2.8%) than in CONT (63.2±0.8%). Plasma membrane integrity was greater in AFPI-0.1 (49.1±4.6%) and AFPI-0.5 (36.6±7.3%) compared with CONT (13.0±4.4%). All AFP groups had a greater percentage of normal sperm (overall mean: 74.3±1.3%) than CONT (65.3±1.9%). There were no significant differences in percentage of sperm with functional membrane (overall mean: 16.1±3.3%), normal acrosome (11.5±4.5%), mitochondrial activity (24.5±6.5%), chromatin condensation (98.8±0.4%), perivitelline membrane binding rate (194.0±44.5 sperm/mm2), and lipoperoxidation (556.7±20.5 TBARS ng±mL−1). In conclusion, the use of AFP, predominantly type I, had potential as a cryoprotectant for ram sperm, increasing sperm cell protection, with no adverse effects on potential fertilization capacity and did not increase reactive oxygen species. This research was funded by FAPERJ, CNPq, and CAPES (Finance Code 001).

Study on embryonic and larval developmental stages of Sucker head Garra gotyla (Gray 1830; Teleostei; Cyprinidae)

SummaryGarra gotyla is an indigenous coldwater fish of the cyprinid family and has wide geographical distribution in India as well as in other countries of Asia and Africa. Induced breeding in G. gotyla was carried out successfully for the first time and an attempt has been made to document developmental stages chronologically from the first minute of fertilization, through all stages of embryonic development until the fifth day post hatching. This experiment was carried out at 22–24°C water temperature at the Directorate of Coldwater Fisheries Research, Bhimtal, India. During the breeding trial, the fertilization rate was observed as 70–75% and hatching rate was 85–90%. The mature fertilized ova were measured as 0.8–1.0 mm in diameter and the perivitelline membrane became thick soon after fertilization and formation of the germ pole. The periods taken for complete developmental stages were recorded; cleavage stage 111 min (min post fertilization (pf)), blastulation stage 580 min (pf), neurulation and segmentation 1250 min (pf) and hatching was completed after 1420 min. The sac fry was measured as 3 mm in length and took almost 3 days for complete absorption of the yolk content. The major structural and differential changes observed are in head, tail, fins, alimentary canal, rudiments of each organ and appearance of melanophore pigmentation in the whole body. The 5-day-old larvae were measured as 6 mm in length with almost every organ fully differentiated. The present study will be utilized for large-scale production of fingerlings for stock enhancement in rivers, lakes and possibilities of genetic improvement and manipulation at the embryonic stage.

Assessing the evidence of ‘infertile’ sea turtle eggs

There is increasing concern about feminization of sea turtle populations resulting from female-biased production of hatchlings due to climate change and selective loss of males from other anthropogenic drivers. Extreme female-biased breeding populations would reduce the likelihood of successful mating and potentially result in high rates of infertile eggs. Infertile eggs are those in which none of the events between sperm penetration of the ovum and syngamy have occurred. Distinguishing between fertile and infertile eggs is challenging, especially in field conditions, and researchers often have relied on physical evidence gathered from unhatched eggs at the end of the incubation period, which likely have experienced tissue decomposition. We argue that infertility in sea turtle eggs can be demonstrated only by the absence of holes caused by sperm penetration of the inner perivitelline membrane; sperm bound between the inner and outer perivitelline membranes; nuclei in the blastodisc; embryonic tissue or membranes in egg contents; and/or the characteristic white spot on the egg exterior. Unhatched eggs can be examined at the end of the incubation period, but we recommend that studies specifically investigating infertility examine at least 20 oviposited eggs each from clutches laid by at least 20 different turtles at the peak of the nesting season.

Modification and Clinical Application of the Inner Perivitelline Membrane Test in Different Avian Species

The aim of this study was to adapt an inner perivitelline membrane (IPVM) test as an interspecies penetration assay for avian spermatozoa. The IPVM of different bird species was evaluated to test the penetrating ability of avian spermatozoa in an intra- and interspecies design. Isolation of the IPVM via acid hydrolysis was tested in pre-incubated chicken eggs and in six other avian species. The separation protocol was modified (time, acid concentration) to facilitate practicability. Separated membranes were evaluated with dark field microscopy for the presence of holes produced by penetrating spermatozoa. In chicken eggs, the influence of different membrane storage conditions was tested. In the penetration assay, the IPVM of chicken eggs was used as a model for fresh and frozen–thawed rooster sperm and for fresh spermatozoa of cockatiels and falcons. Results demonstrated that the time of egg-incubation had a significantly negative influence on the isolation ability of the IPVM (p < 0.0001). IPVM-separation was successful for a maximum of two days after preincubation. In the experiments with eggs from other avian species, results were heterogenous: there was no isolation in geese and cockatiels, 20% in the European kestrel, and 40% in pheasant, quail, and duck. In the penetration assay, holes were found in 100% of the IPVM of chicken eggs after incubation with native and frozen–thawed rooster semen and in 10% with fresh cockatiel semen. Falcon spermatozoa failed to produce visible holes. In conclusion, the IPVM of chicken eggs seems to be unsuitable to establish a functional sperm assay in other species tested but is suitable for quality evaluation of cryopreserved rooster sperm.

Sperm-binding to the perivitelline membrane of chicken egg yolk as a functional test for sperm evaluation in dogs

Durante a fecundação, os espermatozoides interagem com a zona pelúcida (ZP) por meio da ligação entre o acrossomo e as proteínas 2 e 3 (ZP2 e ZP3). A membrana perivitelínica da gema de ovo de galinhas é homóloga à ZP3 de mamíferos, possibilitando a ligação espermática de diversas espécies. Este trabalho padronizou e avaliou a eficiência do teste de ligação espermática à membrana perivitelínica da gema de ovo de galinhas como avaliação funcional do sêmen de cães. Para tal, foram utilizadas nove amostras seminais previamente criopreservadas. Cada amostra foi dividida em duas alíquotas: a primeira foi mantida em banho-maria à 37ºC (vivos) e a segunda submetida a choque térmico com o intuito de induzir dano celular (mortos). As duas alíquotas foram misturadas, correspondendo a 0, 25, 50, 75 e 100% de células viáveis. As amostras foram avaliadas quanto ao número de espermatozoides ligados à membrana perivitelínica por meio da análise computadorizada da motilidade (CASA) ou microscopia convencional. Ademais, as amostras foram avaliadas quanto à motilidade espermática, integridade das membranas acrossomal e plasmática e atividade mitocondrial espermática. O teste de ligação espermática à membrana perivitelínica de ovos de galinha foi considerado um teste de análise seminal exequível tanto para avaliar a capacidade fecundante dos espermatozoides como atributos seminais gerais, especialmente quando realizado em microscopia convencional, expandindo sua praticidade para a maioria dos laboratórios de análise de sêmen canino.

EMBRYONIC DEVELOPMENTAL ANOMALY IDENTIFICATION OF GIANT MIMI-MINTUNO (Tachupleus gigas) DURING ARTIFICIAL INCUBATION PERIOD IN THE VIAL BOTTLES

This study aims to reveal the phenomenon of presence / absence of anomalies in the early development of Mimi-giant mintuno (Tachypleus gigas) during artificial incubation in the vial bottles. Samples 5 eggs are fertilized incorporated into transparent 50 bottles and vials each filled with clear sea water medium. Embryonic stages (instars) hatch, bottles marked, then dumped seawater medium was replaced with 4% formalin solution and glycerin amount of 5% by volume, up to ¾ of the total volume of the vial bottles. Standard stages of giant Mimi-mintuno embryonic normal development Mimi-mintuno according to Itow (1988). The description type of anomalies contained in the post-hatching embryo development. The observations are documented in the form of stereo-microphotograph. The results showed that there are forms anomalies: (a). Delayed development, the structure of the body is not perfect; (b). In observation of the embryo hatches, open shell and egg perivitelline membrane has been opened but delayed development, the structure of the body is not perfect; (c). Embryos after hatched perfectly, abnormalities of morphologic structure such as abnormal protrusion on the dorsal carapace part found. Keywords: Tachypleus gigas, artificially, incubation