A routine use of boar sexed semen is far from being a reality due to many limiting factors among which is the long sorting time necessary to obtain the adequate number of sexed spermatozoa for artificial insemination and the high susceptibility to damages induced by cryopreservation. The aim of this study was to evaluate the modification induced by 24-26 h storage on sorted boar spermatozoa on the basis of their viability, acrosome status, Hsp70 presence, and in vitro fertilizing ability. The percentage of viable cells, according to SYBR green/PI staining, was negatively affected (P < 0.05) by sorting procedure. Moreover, liquid storage significantly (P < 0.05) reduced membrane integrity of sorted spermatozoa as compared to all the other groups. Neither sorting nor storage influenced the percentage of live cells with reacted acrosome, according to FITC-PNA/PI staining. Sorted samples, after 24-26 h storage, were characterized by an increase (P < 0.05) of sperm cells negative for Hsp70, as observed by immunofluorescence, and by a decrease (P < 0.05) in Hsp70 content, as evidenced by western blot. While sorting procedure did not adversely affect both penetration rate and total efficiency of fertilization, these parameters were negatively (P < 0.05) influenced by storage after sorting. In order to minimize damages that compromise fertility and function of sex-sorted boar spermatozoa, the mechanisms by which sorting and liquid storage cause these injures require further study.

In pig production, artificial insemination is widely carried out and the use of fresh diluted semen is predominant. For this reason, there are increasing interests in developing new extenders and in establishing the optimal storage conditions for diluted spermatozoa. In the last few decades, we utilised a homemade diluent (swine fertilisation medium (SFM)) for spermatozoa manipulation and biotechnological application as the production of transgenic pigs utilising the sperm-mediated gene transfer technique. The purpose of the present study is therefore to analyse the ability of SFM, in comparison to four commercial extenders, in preserving the quality of diluted boar semen stored at 16.5°C till 15 days. We utilised some of the main predictive tests as objectively measured motility, acrosome and sperm membrane integrity, high mitochondrial membrane potential and pH. Based on our in vitro study, SFM could be declared as a good long-term extender, able to preserve spermatozoa quality as well as Androhep Enduraguard for up to 6 to 9 days and more.

A simple and efficient method for producing multitransgenic animals is required for medical and veterinary applications. Sperm-mediated gene transfer (SMGT) is an effective method for introducing multiple genes into pigs (Sus, Sus scrofa). The major benefits of this technique are the high efficiency, low cost, and ease of use compared with that of other methods: Sperm-mediated gene transfer does not require embryo handling or expensive equipment. The aim of this study was to investigate the influence of SMGT treatment and exogenous DNA uptake on sperm quality. Even after a coincubation with a 20-fold larger amount (100μg/mL) of DNA than usual (5μg/mL), sperm quality parameters were not significantly affected, confirming the hypothesis that the SMGT protocol itself or the amount of bound DNA do not compromise the possibility of an extended employment of SMGT. More importantly, we found that semen used for in vitro fertilization 24h after DNA uptake gave good cleavage (60% vs. 58%, treated vs. control) and developmental rates definitely positive (41% vs. 48%, treated vs. control). These good results are connected to a competitive efficiency of transformation (62%) due to the numerous improvements in SMGT technique. We demonstrate that SMGT-treated spermatozoa retain good quality and fertilization potential for at least 24h, expanding the possibility to apply transgenesis in field conditions in swine, where the greatest hurdles are fertilization timing and plain procedure.

Little information is available on the quality of stallion spermatozoa after sex sorting. The objectives of the present study were to assess the quality of sex-sorted stallion spermatozoa and determine its fertilizing ability after hysteroscopic low dose insemination. Ejaculates from four stallions were collected and sorted by a MoFlo SX flow cytometer/sperm sorter. Before and after sorting, spermatozoa were evaluated for motility by Computer Assisted Sperm Analysis, viability (SYBR 14-propidium iodide), mitochondrial function (JC-1) and acrosomal status (fluorescein isothiocyanate Pisum sativum agglutinin conjugated). A fertility trial was carried out on four mares (seven oestrous cycles) by hysteroscopic insemination, depositing 5 x 10(6) X-bearing spermatozoa. Sex sorting resulted in a significant decrease (p < 0.001) in all motility characteristics. Sperm viability and percentage of spermatozoa with functional mitochondria were not affected by the sorting process, while the percentage of reacted spermatozoa was higher (p < 0.01) for non-sorted than sorted spermatozoa. Pregnancy rate was 28.6% (2/7) after low dose hysteroscopic insemination. Only one pregnancy was carried to term with the birth of a healthy filly. In conclusion, despite the reduction in sperm motility, sex sorting did not impair stallion sperm viability and mitochondrial activity immediately post-thaw; moreover, the sexed spermatozoa retained the ability to fertilize in vivo.

Leptin is an important hormone regulating nutritional status in humans and animals. Its most relevant activity is at the hypothalamic level, where it modulates food behavior, thermogenesis, and secretion of several pituitary hormones. The exact mechanisms underlying these processes are unclear. The purpose of this study was to verify whether leptin could modulate growth hormone (GH) and prolactin (PRL) secretion acting directly on bovine pituitary cells. Adenohypophyseal explants were cultured with different concentrations of leptin (50, 250, and 500 ng/mL); GH and PRL concentrations in culture media were determined by RIA. On tissues treated with 250 ng/mL of leptin, GH and PRL mRNA, as well as protein content, were estimated by reverse transcription-PCR and Western immunoblotting, respectively. Concentrations of GH in culture media containing 250 and 500 ng/mL of leptin were significantly higher than in controls: 1,063.5 +/- 141.2 (mean +/- SEM) and 1,018.8 +/- 88.4 vs. 748.9 +/- 74.0 ng/mg of tissue, respectively, after 1 h of treatment. Prolactin concentrations were significantly higher in culture media containing 50, 250, and 500 ng/mL of leptin than in controls after 2 h of treatment (547.1 +/- 50.3, 547.5 +/- 58.8, and 577.0 +/- 63.7 vs. 406.8 +/- 43.9 ng/mg of tissue, respectively). Tissues cultured with 250 ng/mL of leptin had significantly higher GH mRNA and lower GH protein content than controls (389.7 +/- 17.9 vs. 289.7 +/- 16.7; 1,601.5 +/- 90.1 vs. 2,212.7 +/- 55.6 arbitrary units, respectively) after 5 h of treatment. In contrast, no significant differences were found for PRL mRNA and protein content, possibly because of a delay in the leptin stimulation of PRL secretion. The results suggest that GH and PRL secretion in bovine pituitary explants can be directly regulated by leptin.

Ghrelin, the endogenous ligand of the growth hormone (GH) secretagogue receptor, is considered a pleiotropic regulator involved in a large array of functions, including control of energy balance, regulation of food intake and, more recently, modulation of the reproductive axis. The present study was aimed at determining the changes in plasma concentrations of acyl-ghrelin in pregnant and lactating sows, with special emphasis on the relationship with the levels of GH, leptin, non-esterified fatty acids (NEFA) and insulin-like growth factor (IGF-1). Blood samples were collected via jugular venipuncture from 22 multiparous sow 30, 60 and 90 days after artificial insemination, 7 and 21 days after farrowing and at first oestrus post-weaning. Plasma concentrations of acyl-ghrelin, leptin, GH and IGF-1 were quantified by validated radioimmunoassay; NEFA were determined using a colorimetric procedure. Plasma acyl ghrelin levels were highest at 30 days of pregnancy and decreased thereafter and during lactation. At the beginning of lactation, GH, IGF-1 and NEFA concentrations significantly increased, while a significant reduction occurred in leptin. In conclusion, ghrelin concentrations in sow maternal circulation does not seem to play an important role in maintaining circulating GH levels during lactation; moreover, ghrelin is not associated with leptin, NEFA and IGF-1 levels.

Leptin is mainly secreted by adipocytes and is implicated in the regulation of metabolic status, feed intake, and body condition. Day length (DL) can affect leptin gene expression and secretion. The aim of the study was to evaluate the effect of DL on gene expression of leptin and leptin receptors in adipose tissue (AT). Four lactating and pregnant Holstein cows were housed in a climate-controlled chamber for 51 d. The first 30 d were used to adapt animals to the new housing conditions. During that period the DL adopted was 12 h light:12 h dark (12:12). The experimental period included 3 different and consecutive phases: 7 d of neutral DL (12:12); 7 d of long DL (18 h light:6 h dark); and 7 d of short DL (6 h light:18 h dark). Subcutaneous AT biopsies were performed at the end of each phase. Prolactin, growth hormone, cortisol, leptin, glucose, nonesterified fatty acids, beta-OH-butyrate, and cholesterol were determined in plasma samples. Abundance of leptin mRNA, and Ob-Ra and Ob-Rb leptin receptor mRNA were determined in AT samples by ribonuclease protection assay. Day length did not affect feed intake or body condition score. Exposure to short DL significantly reduced milk yield (13.1 +/- 2.2 vs. 15.8 +/- 1.7 and 16.0 +/- 2.0 kg/d for short vs. neutral and long DL, respectively). Plasma leptin, growth hormone, cortisol, nonesterified fatty acids, beta-OH-butyrate, and glucose were not affected by DL; cholesterol was lowest under short DL (3.93 +/- 0.38 vs. 4.36 +/- 0.39 and 4.07 +/- 0.38 mmol/L for short vs. neutral and long DL, respectively). Prolactin increased under long DL (134.82 +/- 16.94 vs. 81.98 +/- 20.25 and 96.16 +/- 0.38 ng/mL for long vs. neutral and short DL, respectively). Gene expression of leptin and its receptors was affected by DL. Leptin mRNA increased under long DL (11.91 +/- 0.84 vs. 7.82 +/- 0.84 and 7.56 +/- 0.84 pg of mRNA/microg of total RNA for long vs. neutral and short DL, respectively). Leptin receptors Ob-Ra and Ob-Rb mRNA were higher under long DL, whereas Ob-Ra and Ob-Rb mRNA were lower under short DL (Ob-Ra: 1.91 +/- 0.41, 2.49 +/- 0.41, and 0.65 +/- 0.41 pg of mRNA/microg of total RNA for neutral, long, and short DL, respectively; Ob-Rb: 5.29 +/- 0.79, 5.98 +/- 0.68, and 2.02 +/- 0.70 pg of mRNA/microg of total RNA for neutral, long, and short DL, respectively). Results of the present study appear to exclude an effect of feed intake and metabolic status on leptin gene expression. A prolactin-mediated effect of photoperiod on AT leptin modulation may be proposed in lactating dairy cows.

P142 Fusarium Mycotoxins affect Proliferation of Pig Granulosa Cells In Vitro G Ranzenigo, F Caloni, F Cremonesi and LJ Spicer Department of Vet Clin Sci, Department of Vet Sci and Techn Food Safety, University of Milan, Milan, Italy, Department of Anim Sci, Oklahoma State University, USA Fusarium mycotoxins, like trichothecenes and zearalenone (ZEA), are common grain and foodstuffs contaminants. Some of these like deoxynivalenol (DON) and T2-toxin (T2) can negatively impact pregnancy success in swine, but evidence for direct ovarian effects of DON and T2 is meager. To evaluate the effects of three mycotoxins, ZEA, DON and T2 on porcine granulosa cell (GC) proliferation, pig GC from small follicles (1–5 mm) were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing control (no mycotoxins) or mycotoxins (at various doses/combinations). Each mycotoxin exhibited a biphasic effect on GC growth: ZEA enhanced serumand IGF-Istimulated cell growth at concentrations < 0.10 lM; DON at < 0.1 lM; T2 at < 0.001 lM, and estradiol mimicked the stimulatory effect of ZEA. Whereas ZEA, DON and T2 doses of >9.4 lM, 1 lM and 0.002 lM, respectively, significantly inhibited IGF-I-induced cell proliferation. Complete block of the IGF-I-induced cell proliferation was observed at >3.3 lM for DON and at >0.05 lM for T2. The highest dose of ZEA tested (i.e., 31 lM) only inhibited cell proliferation by 31%. T2 also significantly inhibited basal cell numbers. Neither ZEA nor DON altered the inhibitory effects of T2 on cell proliferation. In conclusion, mycotoxins have direct dosedependent effects on granulosa cell proliferation.

Several reliable methods to produce transgenic animals utilize the male genome. After penetration into oocyte, sperm DNA undergoes dramatic conformational changes that could represent a great opportunity for exogenous DNA to be integrated in the zygote genome. Among the enzymes responsible for sperm remodeling, a nuclease could be involved. The presence of a DNase I in oocytes has not been much investigated. To date, an immunolocalization of DNase I has been reported only in rat immature oocytes and the presence of nuclease activities has been shown in avian oocytes. The present study was conducted to verify whether a DNase-I like activity is present in MII mature pig oocytes. To do this, oocyte extracts were assessed for nuclease activity by a plasmid degradation assay and by zymography; these analyses evidenced a 33 kDa, Ca2+/Mg2+ dependent DNase I-like activity that was inhibited by Zn2+. A further identification of DNase I was achieved by Western blot, immunofluorescence and RT-PCR experiments. Moreover, the presence of the enzyme activity was confirmed by the rapid degradation of exogenous DNA microinjected into the ooplasm. Finally, the exogenous DNA transferred to oocyte by spermatozoa during sperm mediated gene transfer in vitro fertilisation protocol seemed to be protected from DNase I degradation and to persist in the ooplasm till 6 h. These results, together with the high efficiency of sperm based transgenesis methods, suggest that the association with spermatozoa protects exogenous DNA from nuclease activities.

New biotechnologies, such as sperm-mediated gene transfer (SMGT), spermatozoa freezing and spermatozoa sorting have improved the possibilities to produce animals with desirable features. The main problem associated with these technologies is the scarce availability of spermatozoa for insemination. The objective of this study was to develop a laparoscopic insemination (LI) technique in gilt that allows the use of low semen doses resulting in high fertilization rates (FR) and minimal distress to the animal; the efficiency of this technique was compared to conventional artificial insemination (AI). Ten gilts were inseminated 36h post hCG treatment near both utero-tubal junctions (UTJ) with 1.5 × 109spermatozoa/5mL per horn and 10 gilts (C) underwent conventional AI. Embryos were collected either at two to four cell stage (LI, n = 5; C, n = 5) for determination of fertilization rate or at day 6 for evaluation of developmental competence (LI, n = 5; C, n = 5). LI gilts showed a slightly higher FR than control animals. In a second trial, 24 gilts underwent LI with varying doses (1.5 × 108, 1.5 × 107, 1 × 107, 5 × 106 or 1 × 106) of semen. Two to four stage embryos were collected and FR was evaluated in each tube. FR obtained with the lowest dose was significantly different from that with other dosages (P < 0.05). Embryos were cultured in vitro to blastocyst stages (percentage of blastocysts: 79.2 ± 3.6%). In a third trial, five gilts were inseminated with semen processed by SMGT technique; both FR (86.1 ± 9.9%) and transgene protein expression were satisfactory. In conclusion, this study shows that LI can be a useful tool for reducing doses of insemination, without affecting the efficiency of fertilization; this technique could have a wide range of biotechnological applications.

The objective of this study was to determine the effects of staining with Hoechst 33342 and of the entire sorting procedure on boar sperm membrane integrity (using Annexin-V/PI), mitochondrial activity (using JC-1/SYBR/PI) and blastocyst development in vitro; the effect of storage at 17 degrees C for 24h prior to Hoechst staining and sorting was also investigated. The Hoechst staining and the whole sorting procedure reduced the percent of live spermatozoa in both fresh (day 0) and stored (day 1) semen, as determined by both assays; nevertheless, there was no increase in live sperm cells showing signs of early damage (Annexin-V positive, propidium negative), whose percentages remained nearly zero. The majority of Annexin-V positive cells were propidium positive, therefore dead. JC-1 staining evidenced a correlation between mitochondrial activity and viability. However, a significant difference between viable sperm cells and sperm cells with active mitochondria was detected in control and stained sperm, whereas almost all viable sorted spermatozoa had active mitochondria. No significant differences in the in vitro produced blastocysts both on day 0 and 1 were observed. In conclusion, despite the damages induced by sorting procedures, semen sorted as fresh or after storage at 17 degrees C can be successfully used for in vitro production of pig embryos.

Multi-gene transgenic pigs would be of benefit for large animal models in medical, agricultural, and pharmaceutical applications; in particular for xenotransplantation, where extensive genetic manipulation of donor pigs is required to make them suitable for organ grafting to humans. We used the sperm mediated gene transfer (SMGT) method to produce with high efficiency multi-gene transgenic pigs using three genes coding for fluorescent proteins: enhanced blue (EBFP), green (EGFP), and red (DsRed2). All three fluorescent proteins were expressed in 171 out of 195 normally developed morula/blastocysts examined at day 6 post insemination (88%). Genomic DNA of 18 piglets born from two litters was screened by PCR, showing that all piglets were transgenic with at least one gene, 7/18 piglets were triple transgenic, 7/18 double transgenic, and 4/18 single transgenic. Fluorescence in situ hybridization (FISH) analysis revealed multiple sites of integration of the transgenes. RNA and protein expression was found in muscle, heart, liver, hair, and peripheral blood mononuclear cells (PBMCs). These results show that SMGT is an effective method for introducing multiple genes into pigs as shown by the simultaneous expression of three fluorescent proteins. (c) 2005 Wiley-Liss, Inc.