Identification and SNP detection for preimplantation active genes and their association with embryo development and male fertility in cattle

Previous studies have focused on identification and characterization of genes involved in early development. However, information on the relationship between sequence variance of these genes and fertility traits is limited. Therefore, this study was conducted to identify single nucleotide polymorphisms in CDH1, DSC2, TJP1, PKP1, COX-2, CD9, GJA1, PLCζ, AKR1B1, N-PAC and EEF1α genes and their association with male fertility traits Non Return Rate (NRR), semen quality traits (volume per ejaculation, concentration, motility and survivality after thawing) and sperm flow cytometry parameters (plasma membrane integrity, acrosome integrity and DNA fragmentation index) and early embryo development. To full fill these objectives, 11 different cattle breeds were used for screening polymorphism. Genomic DNA of sperm of 310 German Holstein bulls was genotyped for the selected candidate genes. Comparative sequencing results revealed polymorphisms in CD9 at pos. 95 (T>C) in exon 9 having no change in amino acid, in PLCζ at pos. 2749 (G>A) in intron 6, in COX-2 two SNPs at pos. 185 (T>C) and pos. 6 (A>T) in intron 5 and 7 respectively, in AKR1B1 at pos. 159 (A>G) in intron 7, in N-PAC at pos. 5 (C>T) in intron 13, in DSC2 seven SNPs at pos. 281 (T>G) in intron 7, pos. 6b (A>G) and pos. 21 (G>A) in intron 9, pos. 206 (G>A) and pos. 328 (T>C) and pos. 341 (C>T) and pos. 378 (T>C) in intron 12, in CDH1 three SNPs at pos. 47 (C>T) in exon 8 having no change in amino acid, and two SNPs at pos. 43 (G>A) and pos. 92 (C>G) in intron 7, in TJP1 at pos. 50 (C>T) in exon 13 having no change in amino acid and in PKP1 at pos. 140 (G>A) in intron 9. Association analysis revealed significant association of the following genes with various traits: DSC2 and TJP1 (PIdentifikation und SNP-Detektion von preimplativ aktiven Genen und ihre Assoziation mit der Embryonalentwicklung und Bullenfruchtbarkeit Vorangegangene Studien legten ihre Schwerpunkte auf die Identifikation und Charakterisierung von Genen die die frühe Entwicklung beeinflussen. Die Erkenntnisse über die Sequenzunterschiede und deren Beziehung zu den Fruchtbarkeitsmerkmalen sind begrenzt. Das Ziel diese Studie war es, SNPs (Einzelbasenaustasch) in den Genen CDH1, DSC2, TJP1, PKP1, COX-2, CD9, GJA1, PLCζ, AKR1B1, N-PAC und EEF1α zu identifizieren und ihre Assoziation mit den männlichen Fruchtbarkeitsmerkmalen Non Return Rate (NRR), Spermaqualität (Volumen je Ejakulation, Konzentration, Motilität und Überlebensfähigkeit) und Spermium-Qualitätsmerkmale (Plasma Membran Integrität, Akrosomen Integrität und DNA Integrität), sowie der frühen Embryoentwicklung zu klären. Zur Detektion der Polymorphismen, wurden die Genotypen von 11 verschiedenen Rinderassen analysiert. Aus dem Sperma von 310 Bullen der Rasse Deutsche Holstein wurde genomische DNA isoliert und für die Auswahl der Kandidatengene genotypisiert. Der Vergleich der Ergebnisse aus der Sequenzierung ermöglichte es, SNPs in den Genen CD9 im Exon 9, bei Pos. 95 (T>C), Diese Polymophismen hatten keinen Einfluss auf die Proteinstrukturen. Weitere SNPs konnten in den Genen PLCζ im Intron 6, Pos. 2749 (G>A), COX-2 im Intron 5, Pos. 185 (T>C) und Intron 7, Pos. 6 (A>T), AKR1B1 im Intron 7, Pos. 159 (A>G), N-PAC im Intron 13, Pos. 5 (C>T), DSC2 im Intron 7, Pos. 281 (T>C), im Intron 9, Pos. 6 (A>G) sowie Pos. 21 (G>A), im Intron 12, Pos. 206 (G>A), Pos. 328 (T>C), Pos. 341 (C>T) sowie Pos. 378 (T>C), CDH1 zwei SNPs im Intron 7, Pos. 43 (G>A) und Pos. 92 (C>G), in TJP1 im Exon 13, bei Pos. 50 (C>T), Diese Polymophismen hatten keinen Einfluss auf die Proteinstrukturen und im Gen PKP1 im Intron 9, Pos. 140 (G>A) detektiert werden. Die Assoziationsanalyse zeigte, dass sich die Gene DSC2 und TJP1 mit dem Volumen der Ejakulation (P<0.05), AKR1B1 und CDH1 mit der Überlebensfähigkeit des Spermas (P<0.05), CD9, AKR1B1, COX2, DSC2 und TJP1 mit der Motilität (P<0.05), sowie CD9, N-PAC, CDH1 und PKP1 mit der Spermiumkonzentration (P<0.05) signifikant assoziieren ließen. Die gefundenen SNPs hatten aber keinen Einfluss auf die NRR und auf Merkmale der Spermien. Bei der abschließende Genotypisierung von Embryonen für drei Gene (DSC2, CDH1 und TJP1) konnte ein signifikanter Effekt (P<0.05) von TJP1 mit dem Merkmal Entwicklung der Embryonen beobachtet werden

Poloxamer 188 exerts a cryoprotective effect on rooster sperm and allows decreasing glycerol concentration in the freezing extender

[EN] Glycerol is the most widely used cryoprotectant for rooster sperm because it declines the mechanical damage to sperm during the freezing process. Despite its high molecular weight and viscosity, which may be cytotoxic, glycerol can cause damage to cells during the cryopreservation process, resulting in less fertility. Poloxamer 188 (P188) is an embryo cryopreservation supplement effective in many species and also for cell lines and plant cells. We tested the suitability of P188 in the cryopreservation of rooster sperm, considering post-thawing motility, abnormalities, membrane functionality (hypo-osmotic swelling test), mitochondrial activity, viability, apoptosis status, reactive oxygen species production, and ATP content after thawing and the fertility and hatchability after AI. We carried out a factorial experiment with glycerol concentrations of 2% glycerol (G2) and 8% glycerol (G8) and P188 concentrations of 0% (P0), 0.1% (P0.1), 0.5% (P0.5), and 1% (P1) as fixed effects, with replicate (seven) as a random effect. Interactions between glycerol and P188 were found, with G2P1 yielding higher quality and fertility. G8P0.5 yielded better in most parameters, however, not reaching G2P1. G2P1 showed significantly higher results for total and progressive motility, kinetic parameters (average path velocity, straight-line velocity, and linearity), membrane functionality, viability, mitochondrial activity, and ATP content and lower apoptosis, dead sperm, and reactive oxygen species production. G2P1 resulted in the highest percentages of fertilized and hatched eggs, with no effects in the hatched eggs ratio. Interestingly, G2 was less efficient in many parameters than G8 when combined with P0 and P0.1, being equivalent to G8 with P0.5 and superior to any G8 treatment as G2P1. In conclusion, P188 could improve rooster semen cryopreservation and allow reduction of glycerol in extenders, with a consequent impact in the poultry industrySIThis research is supported by a research grant of the University of Tabriz (number 5782

Type III antifreeze protein (AFP) improves the post-thaw quality and in vivo fertility of rooster spermatozoa

[EN] Antifreeze proteins (AFP) have the potential for improving sperm cryopreservation. We have applied Type III antifreeze protein (AFP3) on the cryopreservation of spermatozoa from broiler breeder roosters, aiming to enhance post-thawing quality and fertility. Semen was extended at 37°C in Lake's extender containing AFP3 at 0.01, 0.1, 1, 5, and 10 µg/mL (no AFP3 as control). Post-thawing sperm assessment included sperm motility (CASA), morphology, membrane functionality by hypoosmotic swelling test (HOST), lipoperoxidation as malondialdehyde (MDA) production, and sperm viability, early apoptosis (phosphatidylserine exposure as annexin V-positive staining in viable spermatozoa), and mitochondrial activity by flow cytometry. Fertility was assessed after artificial insemination (30 hens/treatment). Total and progressive motility, membrane functionality, and mitochondrial activity increased in 0.1 and 1 µg/mL AFP, compared to control and other concentrations, whereas apoptosis was significantly lower. VAP, VSL, and viability were significantly higher for 1 µg/mL AFP3 than with the other treatments except for 0.1 µg/mL (which was not always significantly different from the control or other concentrations), and with abnormal forms being significantly lower. The proportion of fertilized and hatched eggs was also higher for 1 µg/mL AFP3, with 0.1 µg/mL also showing significantly higher results than the control, and no differences with other concentrations). In conclusion, 1 µg/mL AFP3 could improve the post-thawing results of rooster spermatozoa frozen in Lake's extender. According to our results, concentrations between 1 and 0.1 µg/mL could be similarly efficientSIThis research was supported by a research grant from the University of Tabriz (number 5782

Estimation of variance components for some production traits of Iranian Holstein dairy cattle using Bayesian and AI-REML methods

The used data set included the records of 131990 Iranian Holstein dairy cattle for first three lactations that were collected from 1981 to 2008 time period by Animal Breeding Center, Iran. The traits which were considered for 305 days of lactation included milk, fat and protein yield and percentages of milk fat and protein. Variance components were estimated using average information restricted maximum likelihood (AI-REML) algorithm using AIREMLF90 software under single trait and repeatability models and Bayesian method by using a Gibbs sampling technique (BAGS) and by MTGSAM and GIBBS3F90 software by same models. The linear statistical models of the analyses included herd-year-season and lactations as fixed effects, age at calving as covariate and animal and permanent environment as random effects. The ranges of heritability estimates for lactations 1 to 3 by animal single and repeatability models using AI-REML and BAGS methods were 0.19 to 0.29, 0.17 to 0.26, 0.20 to 0.25, 0.21 to 0.25 and 0.19 to 0.35 for milk, fat and protein yield and percentage of milk fat and protein, respectively. Repeatability estimates by using BAGS method were 0.44, 0.35, 0.43 and by AI-REML method the values were 0.43, 0.34, 0.39 for milk, fat and protein yield, respectively. The results showed that estimated genetic parameter values by AI-REML analyses for all traits and lactations in both models were smaller than BAGS method. In addition, estimated heritability values for later lactations were lower in comparison with the first lactation

Estimation of genetic parameters for productive and reproductive traits in Esfahan native chickens

Abstract The main aim of this research was to estimate the genetic and phenotypic parameters for productive and reproductive traits of Esfahan native chickens. Traits included body weights at hatch (BW1), 8 weeks of age (BW8), 12 weeks of age (BW12), and at sexual maturity (WSM)], age at sex maturity (ASM), egg number (EN), average egg weight (AEW) in the first 12 weeks of production, and egg production intensity (Eint). Data were collected over 13 generations (during 1998 to 2011) at the breeding center of Esfahan native chickens in Iran. Genetic parameters were estimated by a (bi)-univariate animal model using the restricted maximum likelihood (REML) procedure. Heritability estimates for body weight at different ages varied from 0.14±0.01 to 0.42±0.01. Estimated heritability for reproductive traits ranged from 0.12±0.01 for Eint to 0.36±0.01 for AEW. Estimates of heritability values were moderate but BW1 and AEW showed higher heritability values. Genetic correlation among body weight traits varied from 0.20±0.03 to 0.82±0.02. Fairly small negative Genetic correlation between body weight traits and egg traits (EN and Eint) was small (in the range of -0.22±0.05 to -0.03±0.03), while they showed positive and moderate genetic correlation with the average egg weight, ranging from 0.11±0.04 to 0.39±0.02. There was a low negative genetic correlation (-0.09±0.02) between egg number and egg weight. Therefore, during simultaneous selection for growth and egg production, probable reduction in egg production due to low reduction in egg number may be compared by increases in egg weight

Different concentrations of cysteamine and ergothioneine improve microscopic and oxidative parameters in ram semen frozen with a soybean lecithin extender

P. 68-73The aim of this study was to evaluate the effects of ergothioneine and cysteamine as antioxidant supplements in a soybean lecithin extender for freezing ram semen. Twenty-four ejaculates were collected from four rams and diluted with extenders (1.5% soybean lecithin, 7% glycerol) containing no supplements (control) and cysteamine or ergothioneine (2, 4, 6 or 8 mM). Motility by CASA, viability, plasma membrane functionality (HOS test), total abnormality, lipid peroxidation, glutathione peroxidase (GPx) activity and capacitation status (CTC staining) were assessed after thawing. Using 6 mM of either antioxidant improved total motility. Cysteamine at 6 mM and ergothioneine at 4 and 6 mM improved viability and reduced lipid peroxidation (malondialdehyde concentration). Both antioxidants improved membrane functionality significantly, except at 8 mM. Progressive motility, kinematic parameters, GPx activity, capacitation status and sperm abnormalities were not influenced by the antioxidant supplements. In conclusion, cysteamine at 6 mM and ergothioneine at 4 or 6 mM seem to improve the post-thawing quality of ram semen cryopreserved in a soybean lecithin extender.S

Effects of Male on Reproductive Behavior of Markhoz Female Goats in Breeding Season

In order to study the effect of male on reproductive behavior of female goats, 88 Markhoz breed goats with initial body weight of 42±2kg, 3.5 year old, and 10 male goats were used for 6 weeks. This study was conducted in two phases. In the first experiment 48 female goats were allocated in three groups, each group included eight animals with 2 replicate on both sides of male goats, respectively, at intervals of 0-5, 10-15 and 25-30 meters. In the second experiment, 24 female goats having CIDR were used in an order similar to that of first group. However, the goats were located only in one side of the males. The third group was the control group which was treated in a separate saloon in two groups, each of them having eight animals in it. Results showed a significant effect of male animal in estrus synchronization, the earlier beginning of the reproductive cycle and fertility in the early reproductive season. The first group showed better results in creation and synchronization of estrus in comparison to CIDR received animals (respectively 116.7 vs. 91.6%)

Effect of monensin, yeast, selenoprotein and sodium selenite supplementation in flushing period on some blood elements and sexuality of newborn lambs in Ghezel ewes

Some dietary ions can facilitate the penetration of certain types of spermatic chromosomes in the layer surrounding the oocyte to change the sex ratio of newborn lambs. To check this, 66 Ghezel ewes were used in six groups (11 in each group): Treatments consisted of group A as a control (basal diet), group B (flushing with barley grain), group C (flushing with barley grain+vitamin E+organic selenium), group D (flushing with barley grain+vitamin E+mineral selenium), group E (flushing with barley grain+monensin) and group F (flushing with barley grain+yeast). Ewes received experimental diets for 5 weeks (two weeks before and three weeks after mating). Magnesium, calcium, sodium and potassium of serum were measured. The results showed that ewes in groups C and D had the lowest level of calcium and magnesium and highest level of sodium and potassium in the time of estrous and 21 days after mating (

Effect of rosemary (Rosmarinus officinalis) extracts and glutathione antioxidants on bull semen quality after cryopreservation

<p>The present study determined the effects of the addition of rosemary extract (ROM), glutathione (GSH), and their combination (ROM + GSH) to freezing extender on the quality of bull semen after cryopreservation. Before cryoperservation, the samples were diluted in a tris-egg yolk (TEY) extender containing 5 mM GSH (treatment I), 5 or 10 g L–1 ROM (treatments II and III), and ROM with GSH (5 mM GSH with 5 or 10 g L–1 of ROM) (treatments IV and V). An extender containing no antioxidants (non-ROM/GSH-treated) served as control group. Kinematic parameters were evaluated by means of a computer-assisted semen analysis (CASA). The viability and membrane integrity of the sperm were assessed using eosin-nigrosin stain and the hypo-osmotic swelling test (HOST) at 0 and 2 h after freezethawing. Lipooxidative parameters, superoxide dismutase, and glutathione peroxidase (GPx) activity were assessed after thawing. Treatment III showed positive effects for total motility (TM) (p &lt; 0.01), average path velocity (VAP) (p &lt; 0.001), viability (p &lt; 0.01) and HOST (p &lt; 0.01); however, lipid peroxidation (LPO) decreased (p &lt; 0.05) and GPx activity increased (p &lt; 0.05) immediately after thawing compared to the control. The TM (p &lt; 0.01), VAP (p &lt; 0.01), viability (p &lt; 0.01), HOST (p &lt; 0.01) decreased in LPO (p &lt; 0.01) and GPx activity (p &lt; 0.05) for treatment V and the viability and GPx activity (p &lt; 0.05) for treatment I were significantly higher than for the control group at 2 h after thawing. It was concluded that the inclusion of ROM and its combination with GSH improves the post-thaw quality of bull semen.</p