EFFECT OF FEED RESTRICTION IN A RABBIT LINE SELECTED FOR GROWTH RATE ON REPRODUCTIVE PERFORMANCE: OVULATION INDUCTION, OOCYTE QUALITY, EMBRYONARY AND FETAL LOSSES

Tesis por compendio[EN] The general aim of this thesis was to reproductively characterize females from a rabbit line selected for growth rate and to evaluate the effect of different nutritional strategies (ad libitum and restricted), in order to improve the reproductive performance. In chapter 1, the influence of maternal and embryonic genotype on prenatal survival and fetal growth was evaluated and contrasted with a maternal line. Prenatal survival, fetal weight and fetal placenta weight were affected by both embryonic and maternal genotype. Nevertheless, no differences were detected either at transcriptomic level in fetal placenta or in progesterone and IGF-I plasma levels in these females. It may be concluded that in rabbit females from paternal lines both embryonic and maternal genotypes are key factors in the reproductive performance of these females. The aim of chapter 2 was to explore the causes of ovulation failures in these rabbit females. Results showed that non-ovulated females presented lower LH plasma concentration as well as higher body weight and leptin and BOHB plasma levels than ovulated females. Thus, ovulation failures in females from line R could be attributed to decreased LH plasma concentrations in these females which may be related with their higher body weight and leptin levels. The following three chapters were focused on improvement of the reproductive performance of these females through a different nutritional strategy: a feed-to-appetite diet of these females after the rearing period and prior to insemination. The initial hypothesis was that the females are submitted to a restricted nutritional regimen which is not enough to cope with their needs during reproduction, causing long-term disturbances of energy balance which leads to the subsequent reproductive problems. Chapter 3 aimed to determine if a feed-to-appetite nutritional strategy would affect the hypothalamus-hypophysis axis and the quality of the produced oocytes, by transcriptomic analysis. While no differences were found in the microarray analysis of the hypothalamus-hypophysis, small differences were detected in the transcript expression analysis in oocytes of a group of genes selected. MSY2 was found to be downregulated in oocytes from restricted females. As a key regulator of maternal RNA transcription and translation, changes in this essential gene could explain some of the reproductive problems of these females. Whether the differences found at oocyte level were inherent at embryonic level and so involved in the drop of fertility was studied in Chapter 4. Although no significant differences were revealed in ovulation, embryo recovery, and implantation rate, higher fetal and gestational losses were found in restricted females, as well as lower fetal growth. Thus, we concluded that the nutritional strategy employed may have an impact on the oocyte (Chapter 3), but we also demonstrated that these changes were inherited by the embryo, and result in disturbances in gestational losses and fetal growth. The final chapter of this thesis was conducted to determine whether these effects on reproductive and metabolic elements were also evident in females following the common semi-intensive farm production system. Although the results obtained showed small variances in NEFAs and BOHB plasma levels, and also in body weight, no differences were detected in global reproductive performance in terms of fertility, prolificacy and productivity. The results obtained established that although differences are found at oocyte level and inherited by embryo and fetus, no improvements are reached with the proposed nutritional strategy in terms of reproductive performance when females selected for growth rate lead a normal semi-intensive production system.[ES] El objetivo general fue la caracterización reproductiva de las hembras de una línea de conejo seleccionada por velocidad de crecimiento y el efecto de distintas estrategias nutricionales (ad libitum y restringidas), con la finalidad de mejorar el rendimiento reproductivo. En el capítulo 1 se evaluó la influencia de los genotipos materno y embrionario en la supervivencia prenatal y crecimiento fetal, entre las hembras de esta línea y una línea maternal. La supervivencia prenatal, peso fetal y el peso de la placenta resultaron afectados por los genotipos tanto embrionario como materno, pero no se detectaron diferencias sobre la placenta fetal a nivel del transcriptoma ni en los niveles de progesterona e IGF-I. Por lo tanto, se puede concluir que en éstas tanto el genotipo embrionario como el materno son factores clave en su rendimiento reproductivo. El objetivo del capítulo 2 fue explorar las causas de los fallos reproductivos en estas hembras. Los resultados mostraron que las hembras que no ovularon presentaban menores niveles de LH, un mayor peso corporal y mayor concentración en sangre de leptinas y BOHB, que aquellas que había ovulado. Por ello, los fallos en ovulación detectados en estas hembras podrían estar relacionados con una reducción en los niveles de LH, consecuencia del mayor peso de estas hembras y de los incrementados niveles de leptinas. Los siguientes capítulos se enfocaron a la mejora del rendimiento reproductivo de estas hembras empleando una estrategia nutricional distinta: la ingesta ad libitum de alimento tras el periodo de crianza hasta el momento del comienzo de su vida reproductiva. La hipótesis de partida fue que estas hembras son sometidas a un régimen nutricional restringido que no es suficiente para satisfacer sus necesidades energéticas durante la reproducción, lo que causa alteraciones en su balance energético que se manifestarían en los problemas reproductivos observados. El capítulo 3 trató de determinar a través de un análisis trasncriptómico si la estrategia nutricional planteada afectaría el eje hipotalámico-hipofisario y la calidad de los ovocitos. Aunque no se encontraron diferencias en el análisis de un micrarray realizado sobre el hipotálamo-hipófisis, sí que se detectaron en la expresión génica de los ovocitos. El transcrito MSY2 mostró una menor expresión en los ovocitos de las hembras restringidas. Este gen es un regulador clave en la maduración ovocitaria, por lo tanto, cambios en la expresión de este gen podrían explicar algunos de los problemas reproductivos de estas hembras. En el capítulo 4 se estudió si las diferencias a nivel ovocitario eran heredadas por el embrión pudiendo causar la baja fertilidad de estas hembras. Aunque no se encontraron diferencias en las tasas de ovulación, recuperación embrionaria e implantación, sí que aparecieron diferencias en las pérdidas fetales y gestacionales, así como un menor crecimiento fetal en los embriones procedentes de hembras con restricción alimentaria. Por ello, concluimos que la estrategia nutricional empleada tiene unas consecuencias en el ovocito (Capítulo 3), y demostramos que estos cambios parecen continuar en el embrión, resultando en alteraciones en pérdidas gestacionales y crecimiento fetal. El capítulo final fue desarrollado para evidenciar si los efectos reproductivos y metabólicos observados en los capítulos previos se manifestaban en las hembras que se encuentran en un sistema de producción tradicional en granja. A pesar de que los resultados mostraron variaciones en los niveles circulantes de NEFAs y BOHB y de peso corporal, no se encontraron diferencias en el rendimiento reproductivo global a nivel de fertilidad, prolificidad y productividad. Los resultados obtenidos parecen indicar que a pesar de las diferencias encontradas a nivel ovocitario, embrionario y fetal, con el régimen nutricional propuesto no se logra alcanzar mejoras en la eficiencia reproductivo de las h[CA] L'objectiu general va ser la caracterització reproductiva de les femelles d'una línia de conill seleccionada per velocitat de creixement i l'efecte de diferents estratègies nutricionals (ad libitum i restringides), amb la finalitat de millorar el rendiment reproductiu. En el capítol 1 s'avaluà la influència dels genotipus matern i embrionari en la supervivència prenatal i el creixement fetal, entre les femelles d'aquesta línia i les de una altra línia maternal. La supervivència prenatal, el pes fetal i el pes de la placenta resultaren afectats pels genotipus embrionari i matern, però no es detectaren diferències en la placenta fetal a nivell de trascriptoma ni en els nivells de progesterona i IGF-I. Per tant, en les femelles de conill seleccionades per velocitat de creixement, tant el genotipus embrionari com el matern són factors clau en el seu rendiment reproductiu. L'objectiu del capítol 2 va ser explorar les causes de les fallades reproductives en aquestes femelles. Les femelles que no ovularen presentaren menor nivells de LH, major pes corporal i major concentració de leptines i BOHB que aquelles que sí que hi havia ovulat. Per això, les fallades d'ovulació detectades en aquestes femelles podrien estar relacionades amb la reducció en els nivells de LH, com a conseqüència del major pes d'aquestes femelles i dels incrementats nivells de leptines. Els capítols següents s'enfocaren a la millora del rendiment reproductiu d'aquestes femelles mitjançant una estratègia nutricional distinta: la ingesta ad libitum d'aliment després del període de criança i fins al moment de l'inici de la vida reproductiva. La hipòtesi de partida fiu que aquestes femelles són sotmeses a un règim nutricional restringit que no és suficient per a satisfer les seves necessitats energètiques durant la reproducció, la qual cosa provoca alteracions en el balanç energètic que podrien manifestar-se en els problemes reproductius observats. El capítol 3 tractà de determinar mitjançant un anàlisi trasncritòmic si la estratègia nutricional plantejada podria afectar l'eix hipotalàmic-hipofisiari i la qualitat dels ovòcits. Metre que no es detectaren diferències en el anàlisi del microarray realitzat en el hipotàlem-hipòfisi, sí que es detectaren en l'expressió gènica del ovòcits. El transcrit MSY2 mostrà una menor expressió en els ovòcits de les femelles restringides. Aquest gen es un regulador clau en la maduració ovocitària, per aquest motiu, canvis en la seva expressió gen podrien explicar alguns dels problemes reproductius de les femelles. En el capítol 4 s'estudià si aquestes diferències a nivell ovocitari eren heretades per l'embrió i podrien causar la baixa fertilitat d'aquestes femelles. Encara que no se trobaren diferències en les taxes d'ovulació, recuperació embrionària i implantació, sí que es trobaren diferències en les pèrdues fetals i gestacionals, així com un menor creixement fetal en els embrions de les femelles provinents d'un règim alimentari restringit. Per això concloíem que l'estratègia nutricional emprada té conseqüències en el ovòcit (Capítol 3), i demostrarem que aquest canvis pareixen continuar en l'embrió, resultant en alteracions en pèrdues gestacionals i creixement fetal. El capítol final fou desenvolupat per evidenciar si els efectes reproductius i metabòlics observats en els capítols previs eren manifestats en les femelles que es troben en un sistema productiu tradicional de granja. Tot i que el resultats mostraren variacions en els nivells circulants de NEFAs i BOHB i pes corporal, no es trobaren diferències en el rendiment reproductiu global, en termes de fertilitat, prolificitat i productivitat. Els resultats obtinguts pareixen indicar que tot i que les diferències trobades a nivell ovocitari i embrionari amb un efecte significatiu en el desenvolupament i creixement fetal, amb el règim nutricional proposat no s'aconsegueix assolir millores enNaturil Alfonso, C. (2016). EFFECT OF FEED RESTRICTION IN A RABBIT LINE SELECTED FOR GROWTH RATE ON REPRODUCTIVE PERFORMANCE: OVULATION INDUCTION, OOCYTE QUALITY, EMBRYONARY AND FETAL LOSSES [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/73065TESISCompendi

The Effect of Triptorelin on Ovulation Rate and Conception Rate in Gilts and the Endocrine Profile in Non-Pregnant and Early pregnant Gilts

Triptorelin is a gonadotropin releasing hormone agonist that has been shown to be effective in weaned sow single fixed time artificial insemination protocols. Administration of triptorelin 96 h after weaning has been shown to be effective in synchronizing sows to be inseminated one time 24 h later without reducing pregnancy rate or the number of pigs born. In the current experiment, 23 crossbred gilts (249 d, 123 kg) were administered an intramuscular injection of PG600. Nine days after PG600 administration, gilts were fed 15 mg of altrenogest once daily for 16 d. Twelve gilts (OVU) were administered 200 μg triptorelin 96 h after the last altrenogest feeding. A single artificial insemination was then performed regardless the expression of estrus 126 h after the last altrenogest feeding. Eleven gilts (CON) were inseminated upon the expression of standing estrus and received a second insemination 24 h later. Blood was collected on d 0 (day of triptorelin administration), 1, and 2 after the administration of triptorelin for the measurement of serum concentrations of estrogen and progesterone by RIA. Estrus detection was performed daily beginning 3 d following the last altrenogest administration and ended 7 d after the last altrenogest administration. Gilts were slaughtered on d 33 when reproductive tracts were collected. Reproductive tracts were evaluated for the number of fetuses in the uterus and the number of CL on the ovary. No differences (P \u3e0.05) were found in pregnancy rate, number of fetuses, number of CL, and the ratio of fetuses to CL in all animals. When non-pregnant gilts were removed from the analysis there was a trend (P=0.08) for the CON group to have a greater ratio of fetuses to CL. Expression of standing estrus was also greater (P\u3c 0.05) in the CON group than OVU group. No differences (P \u3e0.05) were found in serum concentrations of estrogen between OVU and CON gilts. The CON group’s expression of estrus occurred around the time when progesterone was at the lowest in the group. The OVU had its lowest serum concentration of progesterone earlier than CON group. This may indicate that the OVU group could have ovulated earlier than the CON group. Data from the first experiment has demonstrated that even though conception rates of fixed timed AI protocols involving GnRH agonists are similar to standard AI, the conception rate versus ovulation rate may show differences in the two protocols. When comparing the ovulation rate with conception rate, our experiment has shown that GnRH agonists, accompanied by a single fixed timed AI, may not be effective in maximizing the number of conceptuses in gilts. Many of the gilts in this experiment failed to become pregnant even though standing estrus was exhibited in the CON group. Anterior pituitary and blood samples from these gilts were collected to determine serum concentrations of insulin like growth factor (IGF) and luteinizing hormone. Anterior pituitaries were also used to determine the expression of IGF receptor, gonadotropin releasing hormone (GnRH) receptor, luteinizing hormone beta (LH-β), IGF binding protein -2, 3, and 5. Anterior pituitaries and serum samples were collected from 15 pregnant and 8 non-pregnant gilts on d 33 after AI. Serum concentrations of estradiol and progesterone and AP concentrations of LH and IGF-1 were determined by RIA. Relative expression of GnRHR, LH-β, IGF-1, IGFBP-2, IGFBP-3, and IGFBP-5 were determined using real time reverse transcriptase PCR. Fold changes in relative expression were determined using the Relative Expression Software Tool. Non-pregnant gilts were assumed to be undergoing the luteal phase of their reproductive cycle. Our data supported this because no differences (P \u3e0.05) were determined in serum concentrations of progesterone or estradiol between non-pregnant and pregnant gilts. Mean AP concentrations of LH were greater (P0.05) between pregnant and nonpregnant gilts. Mean relative expression of LH-β was .8 fold lower (P\u3c 0.05) and IGFBP- 2 tended to be 0.8 fold lower (P=0.095) in pregnant gilts compared to non-pregnant gilts. No differences were found (P \u3e0.05) between pregnant and non-pregnant gilts in relative expression of AP GnRHR, IGF-1, IGFBP-3, and IGFBP-5. Data from the second experiment have shown that changes in multiple endocrine factors, such as LH and the IGF system, may play a crucial part in maintaining early pregnancy but further investigation is needed

Insights from Animal Reproduction

The chapters in this volume of "Insights from Animal Reproduction" address several, particular hot topics in the field of reproduction. The book begins with a comprehensive overview of the cryopreservation of sheep-produced embryos. The following chapter revises the assisted reproductive techniques available for South American wild mammals. Chapter 3 presents the technical procedures necessary to produce transgenic goats. Chapter 4 provides a comprehensive revision of the major molecular determinants of litter size in prolific species. Chapter 5 examines the germ cell determinant transmission, segregation, and function using the zebrafish as a model for germ cell specification in the embryo. Chapter 6 summarizes the current understanding of the molecular and cellular mechanisms regulating the early stages of folliculogenesis. Chapter 7 examines the sperm motility regulatory proteins as a tool to enhance sperm quality in cryopreservation processes. Chapter 8 discusses contemporary knowledge on the effects of extremely low frequency magnetic fields (ELF-MF) on male reproductive function in rodents. Chapter 9 highlights the importance of the cytogenetic evaluation in searching for causes of infertility of phenotypically normal animals, as well as individuals with an abnormal sex development. The last chapter provides evidence that other uterine diseases may be hidden behind the clinical diagnosis of pyometra that in some case may have a poor outcome

Study of failures in a rabbit line selected for growth rate

[EN] Selection for growth rate is negatively related with reproductive fitness. The aim of this work was to analyse the causes of fertility failure in rabbit does selected for growth rate and characterised for reproductive deficiencies (line R). In the experiment, 82 does were divided into 2 groups: naturally mated (NM) and artificially inseminated (AI), to relate luteinizing hormone (LH) concentration with ovulation induction and pregnancy rate by laparoscopic determination. Additionally, in 38 of these females ovulation rate and metabolites determination (leptin, NEFA, BOHB and glucose) were analysed and perirenal fat thickness measurement and live body weight (LBW) determined. The results showed that all ovulated does (both NM and AI) presented higher concentrations of LH than non-ovulated females. In addition, non-ovulated females showed high levels of leptin and BOHB, as well as LBW. Females from line R have an inherit reduced fertility due to ovulation failure as a consequence of a reduction in LH release, which could be explained by a heavier body weight and higher leptin concentrations.This work was supported by the Spanish Research Project AGL2011-30170-C02-01 (CICYT). Carmen Naturil-Alfonso was supported by a research grant from the Education Ministry of the Valencian Regional Government (programme VALi+d. ACIF/2013/296). English text version was revised by N. Macowan English Language Service.Naturil Alfonso, C.; Lavara García, R.; Millán, P.; Rebollar, P.; Vicente Antón, JS.; Marco Jiménez, F. (2016). Study of failures in a rabbit line selected for growth rate. World Rabbit Science. 24(1):47-53. https://doi.org/10.4995/wrs.2016.4016SWORD475324

Recipient maternal genotypes improved the litter size components of a paternal line involved in a MOET programme in rabbits

[EN] An essential factor in the success of multiple ovulation and embryo transfer programmes (MOET) in any species is the selection of the recipient females. In rabbit there is a notable lack of studies on the effects of recipient genotype on postnatal growth. The aim of this study was to evaluate the effects of recipient maternal genotypes on litter size components within a MOET programme applied to a commercial paternal line that appears to have exhausted its selection programme after 37 generations. The experiment was designed using 13 nulliparous donors from the R line (paternal line) to produce 453 embryos, which were transferred to two recipient maternal genotypes (A and V lines) and the own donor paternal line (R line). Litter size components and pre-and postnatal body mass of kits were evaluated. The differences between the genetic groups of recipients were estimated using a general linear model applying Bayesian analysis. The results showed that maternal lines have a high capacity to implant the embryos, maintain the pregnancy and present a favourable environment for embryo development compared to the R line. Specifically, A line dams showed the highest prenatal survival, total born and number born alive without effects on growth traits. The present study demonstrated the applicability of a MOET programme based on maternal ability recipients to improve the number of kits per cycle. Therefore, to allow the genetic improvement programme of meat rabbits to continue using the R line, we recommended applying for a MOET programme as a routine procedure.This research was supported by the projects: Spanish Research project AGL2014-53405-C2-1-P Interministerial Committee on Science and TechnologyRagab, M.; Valdés-Hernández, J.; Vicente Antón, JS.; Marco-Jiménez, F. (2022). Recipient maternal genotypes improved the litter size components of a paternal line involved in a MOET programme in rabbits. Italian Journal of Animal Science. 21(1):1584-1592. https://doi.org/10.1080/1828051X.2022.21421671584159221

Characterization of KNDy Neuronal Activity in Gilts: Distribution and Effect of A Progestin

Puberty is a process that incorporates a large array of both external factors and internal signals. The endocrinology of puberty has been studied in a number of mammalian species and typically depends upon an increase in hypothalamic secretion of gonadotropin releasing hormone (GnRH). Neural inputs regulating GnRH secretion during the pubertal process are not completely understood. In the past decade, attention has focused on the role for a particular set of neurons located in the arcuate nucleus (ARC) of the hypothalamus. They have been named KNDy neurons because they coexpress kisspeptin, neurokinin B (NKB) and dynorphin and have been shown to stimulate (kisspeptin, NKB) or inhibit (dynorphin) GnRH and luteinizing hormone (LH) secretion. While KNDy neurons have been studied extensively in primates, rodents, and sheep, almost nothing is known about this system in porcine, a species of significant agricultural importance. Our studies describe an initial foray into characterizing this system in the porcine hypothalamus. We first determined that, similar to other species, kisspeptin was expressed in the ARC and that NKB and kisspeptin were coexpressed in this region to a very high extent. We then examined the distribution of receptors for NKB (NK3R) and found them to be expressed in the preoptic area (POA) and several areas of the hypothalamus, including the paraventricular nucleus (PVN) and retrochiasmatic area (RCh). However, there was no evidence of NK3R expression in the ARC. The NK3R-positive cells found in the POA did not co-localize with GnRH but expressed close contacts with GnRH neurons. We then used gilts that were ovariectomized (OVX) or OVX and treated with Altrenogest (OVXA), a progestin, to test the hypothesis that Altrenogest would suppress kisspeptin and NKB immunopositive cell numbers in the ARC and NK3R-positive cell numbers throughout the hypothalamus. Surprisingly, kisspeptin containing cell numbers tended to be increased (p = 0.09) in in OVXA gilts (n = 6) compared to their OVX, untreated counterparts (n = 6). In addition, there was no significant change in NKB-positive cell numbers in response to Altrenogest. Subsequent analysis determined a tendency (p = 0.08) for a decrease in the percentage of kisspeptin neurons expressing c-Fos, a marker of neural activation, in OVXA versus OVX gilts. There was no effect of Altrenogest on NK3R-positive cell numbers in any area. These studies mark one of the first investigations into the KNDy system in swine. Many of the characteristics examined here are similar to that previously reported for other mammalian species, with the notable exception of an absence of NK3R expression within the ARC. Treatment with a progestin did not suppress kisspeptin or NKB expression but did tend to reduce activity of kisspeptin neurons. These results suggest that, similar to other species, KNDy neurons likely play an important role in regulating reproduction in swine

Impaired Fertility in Transgenic Mice Overexpressing Betacellulin.

Impaired fertility in transgenic mice overexpressing Betacellulin Peptide growth factors regulate many cellular functions by autocrine, paracrine, juxtacrine or endocrine mechanisms. The epidermal growth factor (EGF)-like peptides are emerging as major players in regulating different aspects of animal and human physiology and pathology. The EGF family elicits essential actions in reproduction. For instance, different Egfr ligands have been shown to be involved in oocyte maturation and ovulation, preimplantational embryonic development, and implantation. Btc, a member of the Egf family, was initially isolated from a mouse insulinoma cell line, and it is expressed in a wide range of tissues in the mouse, with particularly high levels in the heart, lung, liver, kidney, pancreas, small intestine, colon, testis, ovary and uterus. Btc was identified as one of the Egfr ligands expressed in the mouse uterus exclusively at the time of implantation and can also participate as a mediator of luteinizing hormone (LH), prostaglandins (PGs) and progesterone receptor (PGR). Mice lacking Btc are viable, fertile and show no overt phenotype, but transgenic mice overexpressing Btc exhibit a whole array of phenotypical alterations. In the present study, Btc transgenic mice were employed to study the effects of increased growth factor levels in female and male reproduction. The observation of relatively ineffective matings involving transgenic females (non-productive matings and reduced litter size) during routine breeding led us to functionally evaluate the different stages of the reproductive process. The reduced fertility of Btc transgenic females could be attributed to one or more reproductive dysfunctions, such as a decreased ovulation, fertilization or implantation. Therefore, we have studied different aspects of Btc transgenic female’s and male’s reproduction, including puberty initiation, ovulation, in vivo and in vitro oocyte maturation, sperm parameters, in vivo and in vitro fertilization, and implantation in order to uncover the reason for their reduced fertility. Successive matings of Btc transgenic males and females mice with wild-type mice revealed a decrease in litter size as compared with litters produced by control matings. However, the interval to the first litter was not significantly different between groups. Litter size development showed a significant difference between Btc transgenic females and controls females. The onset of puberty occurred essentially at the same age in transgenic and non-transgenic females. The implantation of the Btc transgenic mice was delayed, but this was not the reason for the litter size reduction, because the mean number of total embryos either attached or recovered from the uterus of transgenic females was already markedly reduced when compared to the number of embryos present in the uterus of control females. For this reason, the explanation must be found in processes taking place before implantation (ovulation or fertilization). We evaluated the number of ovulated oocytes and observed that this parameter did not differ between the two genotypes, however, we observed a statistically significant reduction in the percentage of fertilized oocytes in transgenic as compared to control females, identifying the reason for the reduction in the litter size. Next, we carried out in vitro maturation of oocytes. The timing of nuclear maturation differed between transgenic and control oocytes. Therefore, we decided to evaluate the in vitro fertilization rate, which turned out to be impaired in the transgenic group. The expression pattern at the cellular level, studied by immunohistochemistry, revealed a high expression of Btc in the transgenic cumulus cells, which could be an explanation for the altered in vivo and in vitro fertilization. Although the fertility of Btc transgenic males appears to be impaired, these animals do not display evident alterations in sperm production. This study provides evidence that Btc overexpression does not negatively affect spermatogenesis, sperm motility, progression and concentration values. Future studies are needed to clarify whether the altered fertilization is in fact caused by the high expression of Btc in the transgenic cumulus cells. Furthermore, experiments involving the overexpression of a non-sheddable form of Btc are underway and will help to clarify the actions of precursor (membrane-bound) versus mature Egfr ligands during oocyte maturation and fertilization

Birth litter sex ratio affects gilt behaviour, endocrine status and reproductive performance

This thesis tested the hypothesis that females that developed in a male biased litter would be masculinised and therefore have impaired reproductive performance and be more aggressive than those born into female biased litters. One of the aims of this thesis was to investigate new tools for selection of gilts into the breeding herd. Current selection criteria are minimal, with genetics, weight, and body conformation as the main determinants. We investigated effects of the sex ratio of a gilt’s birth litter, as in other litter bearing species it affects reproduction, behaviour, and physiology. One of the mechanisms that may be involved is the transfer of androgens from male to female fetuses during development which can occur either to neighbouring fetuses or to the whole litter. Therefore, females from male biased litters may have impaired reproduction and be more aggressive due to masculinisation during fetal development. A literature review found evidence that this phenomenon may affect pigs. We defined a male biased litter having ≥60% males (including stillborns) and female biased as ≥60% female. We examined sex ratio effects on induction of ovulation prior to puberty, and the endocrine milieu before and during natural oestrus. We also investigated behaviour by applying anxiety and aggression tests. Phenotypic differences were also assessed by measuring the anogenital distance (AGD; distance from anus to the vulva), which is commonly elongated in females from male biased litters. Interestingly, we found, in contrast to findings from other species, that the AGD of gilts from female biased litters was longer than those from male biased litters at 16 weeks of age. The anogenital distance at 21 weeks of age was then employed in a commercial study to examine associations with gilt fertility through two parities. Gilts with longer anogenital distances reached puberty younger, were more likely to be mated, and had a higher total born alive litter size. Based on our earlier data, we suggest these gilts were likely from female biased litters. In our behavioural studies we found that gilts from male biased litters were bolder as they had a faster emergence time in an arena test, but they also showed trends towards increased aggression with increased scratch scores around weaning and greater likelihood to fight in a resident intruder test. We also found that when stimulated with exogenous gonadotrophins at 18 weeks of age, gilts from male biased litters has a higher ovulation rate. During second oestrus, gilts from male biased litters had impaired ovulatory luteinising hormone surges but no change in tonic luteinising hormone. These results highlight that gilts from male and female biased litters have different reproductive responses. Overall, our research shows that gilts from male biased litters are different to gilts from female biased litters in reproduction, behaviour, and physiology. Currently, it appears that gilts from female biased litters, or those with above average anogenital distances, are better suited to the breeding herd but further commercial work is required to assess the effect that this new selection tool has on overall performance.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Animal and Veterinary Sciences, 201