Master of Science / Department of Animal Sciences and Industry / John M. Gonzalez / Pigs fed ractopamine-HCl (RAC) are more prone to fatigue and exhaustion when improperly handled. Wireless electromyography (EMG) can be used to directly measure median power frequency (MdPF) and root mean square (RMS) as indicators of action potential conduction velocity and muscle fiber recruitment, respectively. The objectives of this study were to determine the effect of RAC on exhaustion, EMG measures, and muscle fiber type characteristics when barrows were subjected to increased levels of activity. Thirty-four barrows were assigned to one of two treatments: a commercial finishing diet containing 0 mg/kg (CON) RAC or a diet formulated to meet the requirements of finishing barrows fed 10 mg/kg RAC (RAC+) for 35 d. After 32 d of feeding, barrows were walked around a circular track at 0.79 m/s until subjective exhausted was reached. Time, distance, and speed were measured. Wireless surface EMG sensors were affixed to the Deltoideus (DT), Triceps brachii lateral head (TLH), Tensor fasciae latae (TFL), and Semitendinosus (ST) muscles. After harvest, samples of each muscle were collected for fiber type, succinate dehydrogenase, and capillary density analysis. Speed was not different (P = 0.82) between treatments, but RAC+ barrows reached subjective exhaustion quicker and covered less distance than CON barrows (P < 0.01). The end-point MdPF was not affected by the RAC diet. The RAC diet did not change end-point RMS values in the DT or TLH; however, the RAC+ barrows tended to have decreased ST and increased TFL end-point RMS values (P < 0.07). The percentage of type I fibers tended to be greater (P = 0.07) in RAC+ barrows, but the RAC diet tended to increase (P = 0.07) size of type I fibers and increase (P = 0.03) the size of type IIA fibers. Succinate dehydrogenase was not different between treatments. The RAC+ barrows had more (P = 0.03) capillaries per fiber than CON barrows. A diet containing RAC contributes to increased onset of subjective exhaustion, possibly due to rapid loss of active muscle fibers and chronic loss of oxidative muscle fibers with no change in muscle metabolism.

Tese de mestrado integrado. Engenharia Química. Faculdade de Engenharia. Universidade do Porto. 2010

A study was conducted to evaluate the use of Leucaena leucocephala leaf meal (LLM) as a protein supplement for pigs. In addition, an evaluation of detoxifying LLM, by sun-drying, water-soaking, or treating with ferrous sulphate (FeS04) solution was undertaken. The research involved two experiments; a growth study and a metabolic study. In the growth study sixteen, 12 weeks old Large White x Landrace pigs (average body weight 22.9 ± 2.12 kg) were fed four experimental rations; a commercial grain-based grower /finisher ration (control); or a ration containing 20% of either sun-dried LLM, water-soaked LLM, or FeS04-treated LLM, replacing the basal diet. There was a significant (P<0.05) decrease in liveweight gain, feed intake and feed conversion efficiency in pigs fed the ration containing sun-dried LLM. Growth rate, feed intake and feed conversion efficiency were not affected by the addition of water-soaked and FeS04-treated LLM to the basal diet. Triiodothyronine (T3) and thyroxine (T4) levels in the blood plasma were not affected by the dietary treatments. However, addition of FeS04-treated LLM to the basal diet significantly (P<0.05) decreased the back fat thickness of the pigs. In the metabolic study, the digestible dry matter (DDM) and digestible CP (DCP) were measured as well as mimosine, 3-hydroxy-4-(1H) pyridone (3,4-DHP) and 2,3DHP output in the faeces and urine. / Addition of water-soaked LLM to the diet significantly (P<0.05) lowered the DDM of the diet, whereas addition of FeS04-treated LLM significantly (P<0.05) reduced the DCP. Sun-drying, water-soaking and treatment of LLM with FeS04 solution, did not enhance the output of mimosine or 3,4-DHP in the urine and faeces. The results suggest that water soaking or treatment with FeS04 solution reduces the antinutritional factors (presumably including mimosine) and therefore improves the nutritional quality of LLM containing diets for pigs.

PhD / The aim of this thesis was to investigate the possibility of integrating frozen-thawed boar semen into reproductive technologies and into commercial production of pigs in Australia. This was to be achieved by establishing a semen freezing and AI regime that was of a standard acceptable to industry, and integrating the resultant frozen-thawed sperm into other reproductive technologies, such as flow cytometric sperm sorting and IVF. Initially, a protocol for freezing and thawing boar semen was established, based on the method described by Westendorf et al. (1975) and attempts were made to modify this protocol to improve the post-thaw sperm quality, as determined by in vitro assessment of motility, acrosome integrity and longevity. First, the egg yolk used in the freezing extenders was investigated, and the chicken yolk was replaced with either duck or quail yolk. It was shown that there was no benefit in substituting yolk from duck or quail for the chicken yolk traditionally used in freezing extender. Second, the effect of seminal plasma addition to the freezing extender, or seminal plasma addition to resuspension medium post-thaw was tested. Incorporating whole seminal plasma into the freezing extender at levels above 50% was found to be detrimental to post-thaw sperm quality. Reducing levels to 20% of the final volume improved acrosome integrity, but adversely affected motility of sperm. However, adding 20% seminal plasma to the resuspension medium used after thawing of boar semen had no significant influence on sperm quality compared with resuspension in medium without seminal plasma. The antioxidant catalase, and the iron chelator desferal added to the freezing extender, did not improve post-thaw sperm quality, nor was any benefit seen with addition of these substrates to the resuspension medium post-thaw. However, the bioactive phospholipid PAF and its regulating enzyme PAF:AH appeared to enhance post-thaw motility and acrosome integrity of sperm, respectively, when added to the semen pre-freezing. Unfortunately, due to the restrictions imposed on rPAF:AH as a research drug, it was not possible to test the in vivo effects at this time. After the in vitro experiments were completed, the in vivo fertility of frozen-thawed sperm was tested using the optimal freezing protocol and a novel technology, enabling non-surgical deep intrauterine insemination of sows. The aim was to establish the lowest possible dose of frozen-thawed sperm that could be used, without compromising fertility. Successful pregnancies were achieved with doses as low as 62.5 x 106 frozen-thawed sperm but the farrowing rates were too low to be practicable on a commercial scale. This is the first report of litters born after insemination of such a low dose of frozen-thawed sperm and using the novel DIU insemination technique. However, it was concluded that a double dose of 250 x 106 frozen-thawed sperm was the minimum dose required for maintaining acceptable fertility. Reduction in sperm numbers to such an extent made it possible to consider non-surgical insemination of sex-sorted, frozen-thawed semen. Previously, pregnancies had been achieved only after surgical insemination of sex-sorted boar sperm, or with DIU insemination of unfrozen sperm, immediately after sex-sorting. The low numbers of sex-sorted sperm available restricted the inseminate dose used here to 50 x106 motile sperm. A litter of 5 piglets was born after a low-dose, DIU insemination of sex-sorted, frozen-thawed sperm. This is the first report of piglets born after insemination with sex-sorted frozen-thawed sperm and non-surgical insemination. The low farrowing rate achieved in this experiment prompted the investigation of integrating sex-sorted, frozen-thawed boar sperm into IVF. Morulae were produced after IVF with sex-sorted, frozen-thawed sperm and successfully transferred using non-surgical techniques. This is the first report of pregnancy achieved with non-surgical transfer of embryos produced after IVF and IVC of IVM oocytes with sex-sorted, frozen-thawed boar sperm. Unfortunately, the pregnancy did not hold, and the embryos were lost prior to Day 32, but PCR of non-transferred embryos confirmed successful pre-selection of sex. Overall, this thesis demonstrated that it is still not economically feasible to incorporate frozen-thawed boar semen into the commercial production of pigs although it has considerable application in breeding programmes. However, the development of novel techniques enabling reduction in sperm dose, and for non-surgical transfer of embryos into recipient sows and incorporation of frozen-thawed semen into these technologies means that progress is being made with the integration of reproductive technologies and frozen-thawed semen into the pig industry.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

The impact of habitually consuming a high protein (HP) diet at the upper limit of the acceptable macronutrient distribution range (AMDR) on kidney health is unknown. The current study was designed to test the hypothesis that long-term consumption of a diet providing 35% of energy as protein will have negative consequences on renal health, as assessed in a pig model. Methods: Adult female, non-pregnant, commercial pigs (Genesus) were randomized to receive either NP (15% energy from protein) or HP (35% energy from protein) isocaloric diets for either 4 or 8 months. Diets contained whole protein sources with an animal: plant ratio of 2:1 in the NP diet to mimic the average Canadian diet. The increased protein in the HP diet was achieved by increasing egg and dairy protein sources. Body composition was measured by dual-energy X-ray absorptiometry. Glomerular volume and kidney fibrosis were evaluated on kidney sections by quantitative image analysis. The inflammatory marker monocyte chemoattractant protein-1 (MCP-1) and the growth factor transforming growth factor beta-1(TGFβ1) were assessed in renal tissue using commercial ELISA kits. Results: Pigs given the HP diet had lower body weights and percentage of body fat. Pigs consuming the HP diet had significantly higher glomerular filtration rates (GFR) and larger kidneys. Renal MCP-1 levels and renal fibrosis also were significantly higher in pigs given the HP diet, while proteinuria and renal TGFβ1 expression did not differ. Conclusion: These findings suggest that, despite the potential benefit of the HP diet on body composition, long-term intakes of protein at the upper limit of the AMDR may compromise renal health in healthy female pigs. / October 2008

Rooting and exploring are two behaviours pigs in the wild spend considerable time doing. When kept in pens with limited access to bedding materials stress can originate and often lead to undesired behaviours. The purpose of this study was to investigate whether environmental enrichment in terms of ropes and an activity ball containing piglet food, can provide an outlet for their natural behaviours and thus reduce undesired behaviours. In total, 141 pigs were used and divided into three groups, control-, activity ball- and rope groups. The observation methods used were 1/0 recording and instantaneous recording with 15 second intervals. In the rope group a significant difference was seen in the interaction with enrichment, as it was higher on day 1. Between the three groups, a decrease in ear-, tail- and equipment biting was seen in the two enriched groups on day 1. On day 3 these difference could only be seen between the control group and the other two groups in equipment biting. However, few data ware collected and so we cannot confirm our hypotheses, concerning whether the environmental enrichments decreased the undesirable behaviours or not. However, most of our findings are in compliance with earlier studies as we among other things found that the rope worked better than the activity ball and that the interest of the enrichments decreased with time.

The commensal intestinal microbiota contributes substantially to intestinal development in the early neonatal period by mechanisms that are not yet elucidated but could contribute to novel strategies to improve intestinal health. A series of gnotobiotic experiments using isolator-reared caesarian section-derived piglets inoculated at 1 d of age with selected bacteria and euthanized at 14 or 15 days of age were performed to investigate intestinal morphology, inflammation and digestive function. In Experiment 1, piglets were maintained germfree (GF), mono-associated with Escherichia coli (EC), mono-associated with Lactobacillus fermentum (LF) or conventionalized with sow feces (CV). Increased (P<0.05) gene expression of Fas ligand (FasL) and tumor necrosis factor (TNF?) in EC and CV as compared to LF and GF pigs coincided with increased apoptotic and proliferative activity. Toll-like receptors (TLR) 2, 4 and 9 were differentially regulated (P<0.05) by colonizing species. In Experiment 2 using the same animals as Exp. 1, increased turnover of brush border enzymes was indicated by reduced (P<0.05) specific activity of aminopeptidase N (APN) and lactase (LPH) and increased expression of APN in CV and EC as compared to GF and LF pigs. Reduced enzyme activity to gene expression ratio corresponded with an in vitro assay of microbial inactivation of APN. In Experiment 3, probiotic Lactobacillus sp., L3777, and Bifidobacteria sp., B5445, did not induce expression of inflammatory cytokines in mono-association but di-association with E. coli increased (P<0.05) inflammatory and anti-inflammatory mediators and resulted in a high rate of sepsis (50%) relative to E. coli mono-association. Induced expression of inflammatory cytokines by commensal bacteria through TLR and other means, appear to play a substantial role in microbially-induced enterocyte turnover. Enterocyte immaturity did not account for reduced enzyme activity associated with inflammation as increased expression of APN in response to microbial colonization was observed, suggesting a host response pathway enabling effective competition with the intestinal microbiota for available peptide nutrients. Probiotic bacteria were relatively benign in mono-association but may have facilitated increased translocation of <i>E. coli</i> in di-association. Gnotobiotic animal models are essential to demonstrate outcomes of host response characterized by communication among numerous cell types, although are of significant technical difficulty.

The progress of studies on genes concerning the development and differentiation of early swine embryos have been delayed by limited paucity material. In order to identify the porcine ESTs associates with promoting its breeding efficiency, a cDNA library and ESTs database from oocytes of high reproductive swine is established. Oocytes were obtained from Duroc pig by superovulation which was performed by Taiwan Livestock Research Institute, Council of Agriculture. Total RNA was isolated from 50 mature oocytes, reverse transcription is then performed, followed by PCR based amplification of the cDNA. The amplified cDNA size ranges from 0.4 to 5 kb. The derived cDNA were ligated to a pCR2.1 vector, and the library has complexities of about 5.26¡Ñ104 independent clones. A total of 320 clones was picked and sequenced. By BLASTx analysis, among the 123 sequences, more than 43.07%¡]53/123¡^ mitochondrial proteins are found, 56.91¢H¡]70/123¡^ of the sequence were homologous to known transcripts from human, mouse, Drosophila. In nucleotide level analysis, 82.11¢H¡]101/123¡^ matched with the mitochondrial, ribosome genes and 17.89¢H¡]22/123¡^matched with other homologous genes by BLASTn. PCR analysis of the oocyte library for specific genes revealed transcripts for genes including homologous genes¡]2 pairs highly abundance and 2 pairs low abundance genes¡^, housekeeping genes¡]ACT£] and G3PDH¡^ and developmental genes¡]NEK2 and ZP1¡^. However, novel genes of swine are supposed to be the candidates for high productive phenotypes of swine. The library is a valuable resource for the isolation of clones representing genes active at the early stage. The ability to construct cDNA expression library from a few cells will allow gene expression analysis from oocyte biopsies and derived by nuclear transfer procedures.

The newly discovered oocyte-derived growth factor BMP15, like GDF9, is a member of the transforming growth factor-£] (TGF-£]) superfamily. To our knowledge, however, the expression and function of BMP15 in ovarian tissues has not yet been studied in the pig. We asked whether the relative abundance (RA) of BMP15 mRNA changes in oocytes and follicular cells during pre-ovulatory period or culture of cumulus-oocyte-complexs (COCs) in vitro. Denuded oocytes, cumulus cells and COCs were isolated from growing and pre-ovulatory follicles. Total RNA was extracted from the cells, and reverse transcriptase polymerase chain (RT-PCR) was carried out using specific oligo-nucleotide primers. Expansion of COCs was firstly observed at 18 h, when this period we found BMP15 mRNA expression obviously. But when we culture of denuded oocytes instead of COCs, BMP15 mRNA didn¡¦t express throughout the period. We also detected GDF9 and BMP15 mRNA in pig embryonic stage and in differentiation stage of pig stem cell. GDF9 mRNA level continued to express after fertilization, but BMP15 mRNA didn¡¦t appear. We also added BMP15 antibody against Expanding of COCs. The present results support the concept that BMP15 is a key mediator of oocyte-enabled cumulus expansion in pig.