First-degree relationships and genotyping errors deciphered by a high-density SNP array in a Duroc × Iberian pig cross.

BACKGROUND: Two individuals with a first-degree relationship share about 50 percent of their alleles. Parent-offspring relationships cannot be homozygous for alternative alleles (genetic exclusion). METHODS: Applying the concept of genetic exclusion to HD arrays typed in animals for experimental purposes or genomic selection allows estimation of the rate of rejection of first-degree relationships as the rate at which two individuals typed for a large number of Single Nucleotide Polymorphisms (SNPs) do not share at least one allele. An Expectation-Maximization algorithm is applied to estimate parentage. In addition, genotyping errors are estimated in true parent-offspring relationships. Samples from nine candidate Duroc sires and 55 Iberian dams producing 214 Duroc × Iberian barrows were typed for the HD porcine Affymetrix array. RESULTS: We were able to establish paternity and maternity of 75 and 85 piglets, respectively. Rate of rejection in true parent-offspring relationships was estimated as 0.000735. This is a lower bound of the genotyping error since rate of rejection depends on allele frequencies. After accounting for allele frequencies, our estimate of the genotyping error is 0.6%. A total of 7,744 SNPs were rejected in five or more true parent-offspring relationships facilitating identification of "problematic" SNPs with inconsistent inheritance in multiple parent-offspring relationships. CONCLUSIONS: This study shows that animal experiments and routine genotyping in genomic selection allow to establish or to verify first-degree relationships as well as to estimate genotyping errors for each batch of animals or experiment.

Addition of cholesterol-loaded cyclodextrins to the thawing extender: effects on boar sperm quality.

The aim of the present study was to evaluate the effect that the addition of cholesterol-loaded cyclodextrins (CLC) to the thawing extender has on the quality of frozen-thawed boar sperm. Pooled semen (n = 5) from three boars was used for the experiments. The semen was cryopreserved with an egg-yolk-based extender, it was diluted after thawing in Beltsville thawing solution (BTS) supplemented with different concentrations of CLC (0, 12.5, 25, 50 or 100 mg/500 × 10(6) sperm), and these samples were incubated at 37°C for 150 min. The following parameters of sperm quality were evaluated 30 and 150 min after incubation: sperm with intact plasma membrane (SIPM; %), sperm with normal acrosomal ridge (NAR; %), total motile sperm (TMS; %), progressively motile sperm (PMS; %) and kinetic parameters. Both SIPM and NAR increased (p < 0.05) when the thawing extender was supplemented with 12.5, 25 and 50 mg CLC/500 × 10(6) sperm. Nevertheless, motility decreased (p < 0.05) when the concentration of CLC exceeded 12.5 mg CLC/500 × 10(6) sperm. In conclusion, our results suggest that the supplementation of thawing extenders with CLC improves sperm viability and reduces acrosome damage after freezing/thawing.

Is sperm freezability related to the post-thaw lipid peroxidation and the formation of reactive oxygen species in boars?

The aim of the present study was to determine whether the levels of reactive oxygen species (ROS) substances production and the levels of lipid peroxidation of the sperm membrane were related to the quality that the ejaculates exhibited after cryopreservation in boars. Ejaculates from 42 healthy boars were used in this study and they were cryopreserved with the lactose-egg yolk extender (LEY). Several sperm quality parameters were assessed by flow cytometry in samples incubated for 30 and 150 min at 37 °C after thawing: the percentage of sperm with intact plasma membrane (SIPM), intracellular reactive oxygen substances production through mean of DCF fluorescence intensity of total sperm (mean-DCF) and the percentage of viable and non-viable sperm containing oxidized BODIPY (VSOB and NVSOB). In addition, the percentages of total motile (TMS) and progressively motile sperm (PMS) were assessed at the same incubation times with a computer-assisted sperm analysis system. The classification of the ejaculates into good or bad freezers was performed through hierarchical cluster analysis from SIPM and TMS at 150 min post-thawing. The ejaculates of those males classified as good freezers exhibited higher (p < 0.05) SPIM, TMS and PMS than the bad freezers, although both groups presented similar (p > 0.05) VSOB, NVSOB and mean-DCF. Therefore, these results show that lipid peroxidation and the amount of reactive oxygen substances in the sperm after cryopreservation are similar between boars classified as good or bad freezers.

Reproductive, endocrine and metabolic feto-maternal features and placental gene expression in a swine breed with obesity/leptin resistance.

The current study was conducted in a swine breed (Iberian pig) with a genotype that predisposed the pig to obesity. The aim of the study was to determine the morphological, metabolomic and endocrine features of early conceptuses and to elucidate how placental gene expression (related to placentation, angiogenesis and fetal nutrition), maternal hormones and the metabolome affect the fetal environment and fetal growth. Conceptus viability and growth were found to be related to maternal endocrine (plasma progesterone levels) and metabolic features (plasma levels of leptin, cholesterol, HDL-c, LDL-c and triglycerides). These features were related to the placental expression of the vascular endothelial growth factor A (VEGFA) and leptin (LEP) genes, the placental efficiency and, thus, the nutrition and the metabolism of the fetus (availability of glucose, triglycerides and cholesterol, as HDL-c). Viability of conceptuses in females with evidence of dyslipidemia (low plasma levels of total cholesterol due to low HDL-c concentration but high levels of triglycerides) was diminished. The availability of nutrients and metabolic substrates to the conceptus was also affected in females with higher fat deposition and evidence of dyslipidemia. In conclusion, the conceptus viability and growth appear to be strongly related to maternal metabolic features and, thus, affected in females with alterations in lipid metabolism.

Prepuberal evolution of plasma leptin levels in gilts of thrifty genotype (Iberian pig) and lean commercial crosses (Large White × Landrace).

Current study aimed to determine possible differences in plasma leptin levels during the prepuberal period and their relationship with the onset of puberty in gilts of obese thrifty genotype (Iberian breed) and lean genotype (Large White × Landrace commercial crosses) reared under similar conditions. Plasma leptin concentration increased linearly during the 7 weeks prior to the day of puberty attainment in both genotypes (P<0.005, r=0.707 for LW × L and P<0.0005, r=0.874 for Iberian gilts). However, leptin levels in the Iberian gilts was higher from the first sample of the experimental period, with females having 16 weeks-old (2.7±0.3 vs 1.7±0.2 ng/ml in LW × L; P<0.001), to the onset of puberty (8.5±0.7 vs 2.8±0.3 ng/ml in LW × L; P<0.005). Thus, the current study reinforces previous data on changes in around puberty and evidences, for the first time, profound differences in prepuberal plasma leptin levels between gilts of obese (Iberian) and lean genotypes (LW×L).

Patterns of corpora lutea growth and progesterone secretion in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig).

The current study aimed to compare luteal function, as measured by corpora lutea dynamics and progesterone secretion, in 10 sows with obesity/leptin resistance genotype (Iberian pig) and 10 females of lean commercial crosses (Large White × Landrace). In all the animals, the oestrous cycle was synchronized with progestagens, and ovulation was induced by exogenous gonadotrophins. Thereafter, number and size of follicles and plasma oestradiol concentration were determined at oestrus detection, and number and size of corpora lutea and progesterone concentration were evaluated from Day 4 to 12 of the cycle. There were no differences between genotypes in follicle population and oestradiol concentration, and ovulation rate (15.2±1.3 in Iberian vs 12.7±1.8 in LWxL sows); however, there was a higher percentage of Iberian than control sows showing luteal cysts (66.7% vs 30%, respectively; p<0.05). In both breeds, both total luteal area and plasma progesterone concentration grew linearly from Day 4 to 8 (p<0.01) and remained more stable between Days 8 and 12, without significant differences between genotypes. In conclusion, current study supports that ovulatory processes and luteal functionality are not the main limiting factors for prolificacy in a pig model of leptin resistance and obesity.

Ovulation rate, embryo mortality and intrauterine growth retardation in obese swine with gene polymorphisms for leptin and melanocortin receptors.

The Mediterranean Iberian pigs are obese genotypes, due to a leptin resistance syndrome related to leptin receptor gene polymorphisms. The Iberian pig is affected by a lower prolificacy when compared to lean breeds, and thus may constitute a good animal model for adverse effects of obesity in reproductive performance. The present study determined possible differences in rates of ovulation and embryo implantation and later incidence of embryo mortality and intrauterine growth retardation (IUGR) in sows of Iberian breed (n = 23) and highly-prolific lean commercial crosses (Large White × Landrace, n = 17) at two critical periods of swine pregnancy: Day 21 (just after achievement of trophoblast attachment) and Day 35 (just after completion of the transition from late embryo to early foetal stage). Two different reproductive performances were identified in the Iberian breed; 58.3% of the females had lower ovulation rates than LWxL (13.2 ± 2.3 vs 22.5 ± 1.6, P < 0.05), but 41.7% had a similar number of ovulations (18.2 ± 3.9). However, those Iberian sows having high ovulatory rates showed a high incidence of regression of corpora lutea and embryo losses between Days 21 and 35, which was not found in Iberian females with low ovulation rates; therefore, the number of viable embryos was similar in both Iberian groups (8.2 ± 1.0 and 8.4 ± 1.0) and lower that in highly-prolific LWxL (14.8 ± 1.8, P < 0.05). At Day 35, a total of 167 conceptuses (73 LWxL and 94 IB) were evaluated for IUGR. The LWxL conceptuses were longer and wider than Iberian (69.5 ± 0.1 and 64.4 ± 0.1 vs 49.9 ± 0.1 and 41.9 ± 0.1 mm, P < 0.0001). However, Iberian conceptuses were heavier than LWxL (107.4 ± 6.6 vs 68.6 ± 2.4 g) due to a lower quantity of fluids and a higher development of the placental tissues in comparison to the embryo itself. In conclusion, current study indicates a bimodal effect of obese genotypes on reproductive performances, either by lowering ovulation rate or by increased embryo losses in the first third of pregnancy.

Advanced onset of puberty in gilts of thrifty genotype (Iberian pig).

The Iberian pig is an autochthonous breed of the Mediterranean area, reared extensively in the central and southern areas of Spain and Portugal and that is known worldwide for the production of Iberian ham. The characteristics of the Iberian ham are related to its abundance of intramuscular fat, owing to the high capacity of the pig to accumulate fat under its skin and between the muscular fibres. This ability to store excess fat enables survival during periods of scarcity and it has been found in other antique animal breeds and even in humans, being named as the thrifty genotype. The reproductive management of the Iberian pig, in spite of a lack of accurate information unlike in other swine breeds, is based on the assumption of lower reproductive precocity and efficiency than the modern commercial breeds. The current study characterized and compared the onset of puberty in gilts of Iberian breed and meat commercial crosses (Large White × Landrace) reared in the same conditions by assessing weekly plasma progesterone profiles. At the end of the experimental period, when the gilts became 180 days old, the percentage of Iberian females that had reached puberty was 72.2%, with a mean age and weight of 160.5 ± 2.6 days and 92.9 ± 4.7 kg, respectively. The percentage of Large White × Landrace cross-breed females reaching puberty at 180 days was only 15% (p < 0.05). The mean age and weight were 165.0 ± 4.1 days and 107.8 ± 2.2 kg (p < 0.05), respectively. Thus, converse to the traditional assumption, onset of puberty was advanced in Iberian gilts. These results are important for the reproductive management of the Iberian pig but, at the same time, may set the basis for future studies on the metabolism-reproduction link and, specifically, on the possible relationship between earlier accumulation of fat and attainment of puberty.

Accuracy of in vivo and ex vivo ultrasonographic evaluation of ovarian follicles and corpora lutea in sows.

Current study determined, in sows, the accuracy of ultrasonography for in vivo (n=8) and ex vivo (n=7) evaluation of corpora lutea (CLs) and follicles > or =1.5mm in size, by comparison with macroscopic findings in sliced ovaries. The accuracy for ex vivo detection of follicles increased with follicle size (P<0.05), being low for 1.5-1.9 mm follicles (65.9%) and higher for > or =6mm follicles (93.3%); differences between ultrasonographic and macroscopic observations were significant only for follicles smaller than 3.9 mm (P<0.05), due to underestimation. Ex vivo observation succeeded to detect presence or absence of CLs in all the ovaries; the efficiency for determining the exact number of CLs being 94.4%. The accuracy for in vivo detection of follicles also increased with follicle size (P<0.05), dropping to values lower than 40% for 1.5-1.9 mm follicles; therefore, there were significant differences between ultrasonographic and macroscopic observations (P<0.05). On the other hand, accuracy remained around 92% for > or =6mm follicles. Ultrasonography was useful again for detecting presence of CLs in all the ovaries; the efficiency for determining CLs number reached 86.7%, due to underestimation in ovaries with higher number of CLs (P<0.05). Overall, there were no significant differences when comparing the accuracy of ex vivo and in vivo scannings for determination neither of the number of follicles in each size-category larger than 1.9 mm nor of the presence of ovulations or of the CLs number in each ovary. In conclusion, the use of ultrasonography allows an accurate detection of the presence and number of CLs and follicles > or =2mm of diameter in sows, without significant differences between in vivo and ex vivo observations.