Raul Sanchez-Sanchez

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.

Diet-Induced Swine Model with Obesity/Leptin Resistance for the Study of Metabolic Syndrome and Type 2 Diabetes

The objective of the present study was to determine the suitability of a swine breed with leptin resistance and predisposition to obesity (the Iberian pig) as model for studies on metabolic syndrome and type 2 diabetes. Thus, six Iberian sows had ad libitum access to food enriched with saturated fat (SFAD group; food consumption was estimated to be 4.5 kg/animal/day) whilst four females acted as controls and were fed with 2 kg/animal/day of a commercial maintenance diet. After three months of differential feeding, SFAD animals developed central obesity, dyslipidemia, insulin resistance and impaired glucose tolerance, and elevated blood pressure; the five parameters associated with the metabolic syndrome. Thus, the current study characterizes the Iberian pig as a robust, amenable, and reliable translational model for studies on nutrition-associated diseases.

Gender-specific early postnatal catch-up growth after intrauterine growth retardation by food restriction in swine with obesity/leptin resistance

The effects of undernutrition during pregnancy on prenatal and postnatal development of the offspring were evaluated in sows with obesity/leptin resistance. Females were fed, from day 35 of pregnancy onwards, a diet fulfilling either 100% (group control, n=10) or 50% of the nutritional requirements (group underfed, n=10). In the control group, maternal body weight increased during pregnancy (P<0.05) while it decreased or remained steady in the underfed group. At days 75 and 100 of gestation, plasma triglycerides were lower but urea levels were higher in restricted than in control sows (P<0.05 for both). Assessment of the offspring indicated that the trunk diameter was always smaller in the restricted group (P<0.01 at day 50, P<0.005 at days 75 and 100 and P<0.0001 at birth) while head measurements were similar through pregnancy, although smaller in the restricted than in the control group at birth (P<0.05). Newborns from restricted sows were also lighter than offspring from control females (P<0.01) and had higher incidence of growth retardation (P<0.01). Afterwards, during lactation, early postnatal growth in restricted piglets was modulated by gender. At weaning, males from restricted sows were still lighter than their control counterparts (P<0.05), while females from control and underfed sows were similar. Thus, the current study indicates a gender-related differential effect in the growth patterns of the piglets, with females from restricted sows evidencing catch-up growth to neutralise prenatal retardation and reaching similar development than control counterparts.

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.

Effect of an obesogenic diet during the juvenile period on growth pattern, fatness and metabolic, cardiovascular and reproductive features of Swine with obesity/leptin resistance.

The objective of this study was to determine, in a female swine model of leptin resistance (Iberian pig), the effect of an obesogenic diet, with high saturated fat content, during the juvenile period, on the appearance of early obesity and its possible effects on metabolic syndrome-related parameters and reproductive features (puberty attainment). Thus, from 130 to 245 days-old, seven Iberian gilts had ad libitum access to food enriched with saturated fat whilst six females acted as controls and had ad libitum access to a commercial maintenance diet. Results showed that a high fat intake-level during the juvenile period induces early obesity with lower body weight and size but a higher body fat-content. Such obesity was related with impairments of glucose regulation predisposing for insulin resistance, but also with an earlier onset of puberty. However, there were no signs of hypertriglyceridemia and hypertension; the gilts diminish their intake level and modify their metabolic features by increasing insulin secretion. In conclusion, Iberian gilts freely eating saturated fat diets during the juvenile period have the prodrome of metabolic syndrome but, during their juvenile period, are still able to develop an adaptive response to the diet.

Reproductive consequences of a reciprocal chromosomal translocation in two Duroc boars used to provide semen for artificial insemination.

Cytogenetic analysis of 58 boars at an artificial insemination (AI) centre revealed the presence of a reciprocal chromosome translocation, rcp(1;11)(q-;p+), in two Duroc boars. Pedigree analysis of these two boars suggested familial transmission of the chromosome rearrangement. The reproductive consequences of this translocation were determined in a herd of sows that had received semen doses from these and other boars. All sows underwent multiple AI, with different groups established retrospectively depending on the percentage of semen doses provided by the carrier boars ([number of carrier boar doses/total number doses provided] x 100): 0%, 25%, 50%, 75%, 100%. The fertility rates (percentage of successful multiple AIs/total multiple AIs) recorded for multiple AI including semen doses from the carrier boars were not significantly different from those recorded when all semen doses were supplied by normal-karyotype boars. A reduction in litter size of 29.38% was observed, however, in litters sired by one of the carrier boars when its participation in multiple AI was 100%. The number of live-born piglets per litter gradually decreased (P<0.05) as the percentage participation in multiple AI (25, 50, or 75%) of the carrier boar increased. In addition, both carrier boars sired some piglets with signs of cleft palate and complex malformations of the front legs; these died soon after birth. In conclusion, the boars carrying the translocation rcp(1;11)(q-;p+) showed reduced reproductive performance.

Early-postnatal changes in adiposity and lipids profile by transgenerational developmental programming in swine with obesity/leptin resistance

Maternal malnutrition during pregnancy, both deficiency and excess, induces changes in the intrauterine environment and the metabolic status of the offspring, playing a key role in the growth, status of fitness/obesity and appearance of metabolic disorders during postnatal life. There is increasing evidence that these effects may not be only limited to the first generation of descendants, the offspring directly exposed to metabolic challenges, but to subsequent generations. This study evaluated, in a swine model of obesity/leptin resistance, the existence and extent of transgenerational developmental programming effects. Pre- and postnatal development, adiposity and metabolic features were assessed in the second generation of piglets, descendant of sows exposed to either undernutrition or overnutrition during pregnancy. The results indicated that these piglets exhibited early-postnatal increases in adiposity and disturbances in lipid profiles compatible with the early prodrome of metabolic syndrome, with liver tissue also displaying evidence of paediatric liver disease. These features indicative of early-life metabolic disorders were more evident in the males that were descended from overfed grandmothers and during the transition from milk to solid feeding. Thus, this study provides evidence supporting transgenerational developmental programming and supports the necessity for the development of strategies for avoiding the current epidemics of childhood overweight and obesity.

Sex and breed-dependent organ development and metabolic responses in foetuses from lean and obese/leptin resistant swine.

The present study aimed to determine the effects of breed and sex on growth patterns and metabolic features of advanced-pregnancy foetuses exposed to the same environmental conditions. Thus, at Day 62 of pregnancy, swine foetuses from an obese breed with leptin resistance (Iberian breed) were compared to lean crossbred foetuses (25% Large White ×25% Landrace ×50% Pietrain). There were differential developmental patterns in foetuses with leptin resistance, mainly a higher relative weight of the brain resembling “brain-sparing effect”. Prioritization of brain growth may be protective for the adequate growth and postnatal survival of the Iberian individuals, an ancient breed reared in extensive semi-feral conditions for centuries. There were also clear sex-related differences in foetal development and metabolism in the Iberian breed. Female Iberian foetuses were similar in size and weight to male littermates but had a significantly higher relative liver to body weight ratio resembling “liver-sparing effect” and a trend for a higher relative intestine to body ratio. Moreover, the availability of triglycerides, cholesterol and IL-6 in female Iberian foetuses was similar to that of lean crossbred foetuses. Overall, these features may favour a better postnatal survival and development of females, the sex more critical for the species survival. These findings set the basis for future translational studies aimed at increasing the knowledge on the interaction between genetic and environmental factors in the early programming of the adult phenotype.

Inclusion of seminal plasma in sperm cryopreservation of Iberian pig

The aim of the present study was to evaluate the inclusion of seminal plasma (SP) in the freezing extender, trying to preserve as much as possible of SP with spermatozoa from Iberian pigs, thus improving the conservation of animal genetic resources of this breed. Experiment 1, evaluated the effect of substituting water with SP as diluent in the freezing media in different proportions (0%, 10%, 25%, 50%, 75% and 100%), over pre-freezing (at 10°C and 5°C) and post-thawing sperm quality. The results showed that over 50% of SP in the extender, significantly decreased sperm quality in comparison to the control sample (0% SP) and the samples with 10% and 25% of SP (P<0.05). No significant differences were found between the control sample and the samples with 10% and 25% SP (P>0.05), but treatment with 25% did not show significant differences between the time of incubation at 37°C after thawing (P>0.05), showing greater sperm quality resistance over time. Experiment 2, evaluated the effect of prolonged incubation period, until 480min (simulating the lifespan of sperm in the female genital tract), of sperm samples with 0%, 10% and 25% of SP. Treatment with 25% of SP maintained better sperm quality over time, compared to control sample. Significant differences were observed especially in the parameters of motility analysis (TMS, total motile spermatozoa; PMS: progressive motility spermatozoa. P<0.05). In Experiment 3, the effect of the presence of SP was evaluated during the thawing process. Although some differences were observed between treatments, these differences were not as clear as the previous experiments. In conclusion, replacement of 25% of the water by SP as diluent in the freezing extender could be considered the maximum percentage of inclusion, without harmful effects to the sperm. In addition, this proportion of SP maintained Iberian sperm quality for longer time when it was present during the freezing and thawing process.

Preovulatory follicle dynamics and ovulatory efficiency in sows with thrifty genotype and leptin resistance due to leptin receptor gene polymorphisms (Iberian pig).

This study compares follicular function and ovulatory efficiency in 20 sows with obesity/leptin resistance genotype (Iberian pig) and 20 females of lean commercial crosses (Large White × Landrace; LW×L). Estrous cycle was synchronized with progestagens; ovulation was induced with eCG and hCG, in half of the females of each group, to determine its effect. In females of both breeds not treated with gonadotropins, the number of follicles larger than 4.9 mm and the estradiol secretion increased throughout the follicular phase (P<0.05); estradiol values were similar at estrus detection (22.5±1.2 vs. 26.5±0.6 pg/ml respectively, for Iberian and LW×L sows). Moreover, ovulation rate was higher in Iberian pigs (15.3±1.3 CLs) than in LW×L (10.2±1.3 CLs; P<0.05), with mean progesterone values being 18.1±0.7 ng/ml in Iberian and 16.8±0.6 ng/ml in LW×L pigs. Thus, the preovulatory follicular growth and the ovulatory efficiency seem not to be the main limiting factors for reproductive efficiency in Iberian swine. The gonadotropins induced a significant increase, when compared to untreated females (P<0.05), in the number of follicles larger than 4.9 mm growing throughout the follicular phase; however, estradiol values at estrus were lower (P<0.05) in both breeds (9.2±0.7 pg/ml in Iberian vs. 8.6±0.8 pg/ml in LW×L), when compared with the nontreated animals, which suggests defective follicular function after gonadotropin stimulation. There were also no differences between genotypes in ovulation rate (15.2±1.3 vs. 12.7±1.8) and progesterone secretion (21.2±0.8 ng/ml in Iberian and 20.9±0.7 ng/ml in LW×L sows) in the treated animals. In conclusion, the current findings indicate that preovulatory follicular growth and ovulatory efficiency are not main limiting factors for prolificacy in a pig model of leptin resistance and obesity.