Genetic Engineering Approaches to Pig Production*

Modern biotechnology promises a number of new applications in animal breeding and production. Although conventional pig breeding has achieved a high level of efficiency and productivity numerous problems have been encountered with animal health and the loss of meat quality. Selection based on phenotypic performance data of individual animals does not take into account the importance of specific genes and their relevance within a complex regulatory system. In most cases it is therefore difficult to trace back the genetic origins of clinically important disorders. The application of genetic engineering techniques in pig production will facilitate diagnosis, improvement of productivity, and animal health by allowing direct genetic manipulation. Attention must be focussed on the physical and genetic analysis of the procine genome. The isolation and characterisation of genes, DNA-markers, polymorphic DNA-fragments, and their chromosomal assignment will be important prerequisites and tools for the elucidation of genetic disorders. Especially the detection of heterozygous carriers of recessive disorders and their elimination from the breeding stock will increase selection accuracy and decrease the generation intervals. But also the rapid and simple detection of infectious diseases, which is sometimes difficult if not impossible at present, will improve animal health and welfare. Although the production of transgenic animals either by DNA-microinjection into zygotes or the use of embryonal stem cells manipulated in vitro is less straightforward than DNA-based diagnosis it will play an important role in the direct manipulation of the porcine genome and genes. Breeding programmes including the use of transgenic livestock have already been developed. There is no doubt that genetic engineering has reached a degree of practical feasibility, allowing it to play an important role in pig breeding in particular and animal production in general.     

Deep Freezing of Boar Semen Packaged in Plastic Bags and Straws

For practical reasons, a large volume (i.e. 5 ml) of frozen boar semen per insemination dose is desirable, but successful freezing has not been achieved, since optimal cooling rates have not yet been established. Post-thaw motility and the acrosome intep'ty of semen from four boars frozen with a programmable freezin machine, in mini-(0.25 ml), maxi-(5 ml) plastic straws and in 10 × 5 cm PVC- or Teflon FEP-plastic bags (0.35 – 0.12 mm thick, 5 ml) was studied. The freezing of the semen was monitored using thermocouples placed in the straws and the bags. The freezing curve started from +5°C, at a rate of −3°C/min, to – 6°C, it was held for 1 min at −6°C, and was followed by further drop to −100°C at a rate of −20°C/min, with subsequent storage in LN₂. The bags had a much shorter freezing point plnteau, compared to the maxi-straws. Post-thaw sperm motility was significantly higher when semen was frozen in mini-straws or in bags than in maxi-straws. The freezing procedure did not cause major acrosomal damages, significantly more normal apical ridges being present in the bags and mini-straws than in the maxi-straws. This in vitro evaluation indicates that the freezing method employed is satisfactory for freezing large volumes of boar semen into plastic bags .     

Distributions of extracellular matrix and carbonic anhydrase-III during bovine palatine ridge development.

The present study describes histological alterations and immunohistochemical distributions of extracellular matrices (ECMs) and the carbonic anhydrase isozyme-III (CA-III) during the period of bovine palatine ridge formation. Morphogenesis of bovine palatine ridges was preceded by epidermal placodes and the mesenchymal condensation (MC). During the early stages of less than 44 cm crown rump length (CRL), fibronectin (FN) was distributed densely in the MC. Strong reactions against type I collagen (C-1) were detected outer to the FN positive site. In the stages of more than 44 cm CRL, FN and C-1 were distributed diffusely in subepithelial mesenchyme. Laminin (LN) and type IV collagen were distributed in the epithelial and endothelial basement membranes (BMs) in all of the stages examined, except in the stage of 7 cm CRL, where LN was not detected only in the BM just beneath the epidermal placode. CA-III was detected in basal epithelial cells except for palatine ridge rudiments in the stages of more than 21 cm CRL. It is suggested that the expressions of LN and CA-III might play a role in the spatial determination of rudiments of bovine fetal palatine ridges.     

A novel HPLC method for glutathione-insulin transhydrogenase assay using fluorescence labeled insulin

The activity of rat liver glutathione-insulin transhydrogenase (GIT) was measured by HPLC. The degradation of fluorescein isothiocyanate-I (FITC-I)-labeled insulin is separated into several peaks, which are bound different amount of FITC-I. We selected mono-fluorescein-thiocarbamylated insulin to estimate the decrease of insulin content and it became possible to assay GIT activity. This novel method was time-saving and simple, and this system could utilize instead of previous method.     

Changes in the concentration of mRNAs for the inhibin subunits in ovarian follicles after administration of gonadotropins to progestin treated ewes.

RNA was extracted from single or small groups of ovine ovarian follicles after treatment of ewes with FSH and/or LH. The content of mRNA for the alpha-inhibin and beta A-inhibin subunits was analyzed by hybridization with specific cDNA probes. All ewes were treated with progestin vaginal pessaries to suppress spontaneous preovulatory follicle maturation and ewes were given three intramuscular injections of gonadotropins at 8-hr intervals starting 24 hr prior to collection of ovaries. In experiment I, both Schering-FSH and NIDDK-oFSH-17 (oFSH) significantly increased alpha- and beta A-inhibin mRNA per ewe in 2-5 mm follicles and tended to increase alpha- and beta A-inhibin mRNA in large (greater than 5 mm) follicles. In experiment II, oFSH and NIDDK-oLH-25 (oLH) were administered in a 2X2 factorial arrangement. Separate administration of oFSH or oLH increased (P less than .05) the alpha-inhibin mRNA concentration in large follicles. alpha-inhibin mRNA concentration in 4-5 mm follicles was also increased by oFSH but was decreased by oLH. Concomitant treatment with oFSH and oLH did not change alpha-inhibin mRNA concentrations from those measured in oFSH treated ewes. In experiment II, beta A mRNA concentrations followed a pattern similar to that of alpha A mRNA, but the differences were not statistically significant. We conclude that, in the ewe, exogenous FSH increases the concentration of inhibin mRNA in the whole follicle. The ability of exogenous oLH to alter expression of the inhibin subunit genes may depend upon the stage of follicle maturation.     

In situ hybridization analysis of ovarian prostaglandin endoperoxide synthase mRNA throughout the periovulatory period of the ewe.

Cellular alterations in level of expression of mRNA encoding for prostaglandin endoperoxide synthase were quantified within ovarian tissues of sheep obtained before, during and after induction of the preovulatory surge of LH and ovulation with LHRH. This was accomplished by isotopic in situ hybridization using a selective cRNA probe to ovine prostaglandin endoperoxide synthase mRNA. A significant elevation in mRNA was detected within the theca interna of the preovulatory follicle at 8, 16 and 24 hr following administration of LHRH. Very close to the time of ovulation (ie., at 24 hr post-LHRH) a marked rise in mRNA was observed in association with epithelial cells covering the apical surface of the follicle. Ovarian cyclooxygenase metabolites of arachidonic acid produced during the ovulatory process in the ewe originate within the thecal layer and germinal epithelium of the follicle destined to ovulate.     

Postovulatory Effect of Intravenous Administration of Lipopolysaccharide (Escherichia coli, O55:B5) on the Endocrine Status of Recently Ovulated Sows

The effects of lipopolysaccharide ( Escherichia coli , O55:B5), administered 18 h after ovulation in the second oestrus after weaning on the hormonal profiles in 14 Swedish cross-bred (Landrace × Yorkshire) multiparous sows were studied. The endotoxin group (E-group) sows were administered with 300 ng/kg of lipopolysaccharide (LPS) whereas the control group (C-group) sows were administered 5 ml of saline intravenously via an indwelling jugular cannula. Blood samples for hormonal analyses were collected from all sows until slaughter. In the E-group, progesterone, cortisol and prostaglandin F_2α metabolite levels increased significantly (p < 0.05) following LPS compared with the C-group. It can be concluded from this study that apart from elevating cortisol and prostaglandin F_2α metabolite, LPS also elevates progesterone levels.