Transgenic rabbits provide a useful biological model for the study of the regulation of mammalian genes. However, transgene integration efficiency has generally been low. Here we present a first attempt to increase the integration rate of exogenous DNA into the rabbit genome, using a double pronuclei microinjection method. Pronuclear stage rabbit embryos were recovered from superovulated NZW females, 19-20 h after hCG injection. About 5 microg/mL of exogenous DNA solution was microinjected either into one pronucleus (single microinjection, SM) or into both pronuclei (double microinjected, DM). The transgene consisted of a 2.5 kb murine whey acidic protein promoter (mWAP), 7.2 kb cDNA of the human clotting factor VIII (hFVIII), and 4.6 kb that of 3' flanking sequences of the mWAP gene. The in vitro survival of DM embryos to the blastocyst stage was lower than that of SM embryos (68 vs. 89%). Similar results were obtained using EGFP as a control gene construct. However, there was no difference in the percentage of embryos that developed into live offspring using DM (25%) vs. SM (26%). The integration frequency of mWAP-hFVIII into the genome of transgenic rabbits was 3.3% (1/30) upon SM and 8.1% (4/49) at DM (p < 0.05). All founders transmitted the transgene to their offspring in a Mendelian fashion. The SM founder female secreted 87.4 microg/mL rhFVIII in milk, with an activity of 0.594 IU/mL. The DM founder female produced 118 microg/mL rhFVIII, with activity values of 18 IU/ mL. This is the first report of transgenic rabbit production using a double microinjection technique. Our preliminary results suggest that this method can increase the efficiency of production of transgenic rabbit founders, giving a higher integration rate than single microinjection.

This study was conducted to investigate the effect of heat stress (i.e., elevated ambient temperature -Ta; 36 °C ± 3 °C) on growth performance, mortality rate, and on some haematological and biochemical parameters in different categories of gender and age of New Zealand White (NZW) rabbits. Animals were divided into two main groups (control and treatment), in each group there were 56 rabbits: adult females (n = 20), adult males (n = 4), growing females (n = 16), and growing males (n = 16). Results revealed that total and daily feed intake, feed conversion ratio, and total and daily gain in body weight for growing NZW rabbits were affected negatively by elevated Ta. Decreases in feed intake led to less protein biosyntheses and less fat deposition, which led to lower body weight gain. These observations were made in growing and adult rabbits of both genders. Analysis showed that red blood cell (RBC) counts showed alterations. Packed cell volume (PCV) (in adult females and males), white blood cell (WBC) counts (in growing females), lymphocytes (in growing males), monocytes (in growing females and adult males), basophils (in growing females and growing and adult males) were significantly (P < 0.05) decreased, and total proteins (TP) (in adult females), glucose (Glu) (in adult females), and calcium (Ca 2+ ) (in growing males and females) were significantly (P < 0.01) lower in the experimental group. Furthermore, elevated Ta increased the mortality rate (MR) in both age groups. The mortality rate was 30.36% for growing and adult rabbits of the experimental group, compared with 7.14% for the control group, and was 25% for adult compared with 34.38% for growing experimental rabbits. Exposure of NZW rabbits of both ages and genders to elevated ambient temperature (36°C ± 3 °C), negatively affected their internal homeostasis which was reflected in their growth rate and various physiological signs.

Effects of a 50 Hz extra-low frequency electromagnetic field (ELF EMF) on in vitro rabbit spermatozoa motility were analyzed, as well as the effect on fertilization rates after insemination. Pooled semen samples and a control were exposed to 50 Hz ELF EMF. The difference of the samples of the test groups G1 and G2 with the control group CG (75.56%) for spermatozoa motility were found to be significant (P < 0.01). Differences were significant (P < 0.01) for curvilinear velocity (VCL) between the test group G3 (122.38 microm/s) and the control group CG (112.02 microm/s). Hormonally stimulated adult (9-12 months) females (n = 140) were inseminated with semen samples from G1, G2, G3 and CG (0.88 x 109 spermatozoa/0.5 mL average insemination portion) immediately after ELF EMF exposure and fertilization (kindling) rates were calculated. For the G2 it was 54.28% data indicate 50 Hz ELF EMF induced alterations of spermatozoa motility and kindling rate in rabbits, therefore influencing fertility.

The aim of this study was to investigate the effect of dietary supplementation of seaweed on the reproductive performance of rabbits. Two trials were performed during this study. In the first trial, semen quality was evaluated in 15 buck rabbits with mean body weight of 4.8090.41 kg and six month of age. In the second trial, prolificacy was determined in 30 artificially inseminated does with a mean body weight of 4.8490.50 kg and five to six months of age. Rabbits in each trial were randomly allocated to one of three dietary seaweed levels; commercial pelleted diet (C), pelleted diet supplemented with 1% seaweed (T 1 ), and pelleted diet supplemented with 2% seaweed (T 2 ). Dietary supplementation of seaweed significantly increased plasma testosterone concentration and improved various sperm motility parameters. Analysis of acrosomal membrane integrity using electron microscopy revealed no significant influences of dietary seaweed supplementation on quality grade (IÁIV) of all tested samples. These results reflected in positive prolificacy response of does artificially inseminated with semen samples pooled from bucks fed on T 2, and offered diet supplemented with 2% seaweed one week prior to their insemination and throughout the gestation period. Feeding diets supplemented with 2% seaweed to doe rabbits improved their kindling rate, litter size, and their offspring ratio. Seaweed supplementation to the diets of rabbits raised under summer conditions had improved their reproductive performance by improving the semen fertility characteristics of bucks and the prolificacy characteristics of does. Hematological and biochemical parameters investigated in this study did not reveal any pathological signs in both rabbit's genders due to dietary seaweed supplementation.

The aim of this study was to verify whether the separation and elimination of the apoptotic fraction in rabbit semen using a MACS technique may improve sperm fertility potential and consequently rabbit kindling rate. Semen samples from 25 New Zealand White (NZW) rabbit males were collected using an artificial vagina and evaluated using the CASA system for concentration and motility. For artificial insemination the best 11 bucks were chosen based on motility parameters. Their ejaculates were mixed to make a heterospermic pool and routinely diluted in a commercial insemination diluent (MiniTüb, Tiefenbach, Germany) at a ratio of 1:6. Diluted heterospermic spermatozoa were filtered through a Sartorius filter to wash out seminal plasma, re-diluted in binding buffer (Annexin V Microbead Kit, Miltenyi Biotec, Germany) at a ratio of 1:3.66 and divided into two groups: an experimental group intended for MACS separation and control group without MACS separation. Then hormonally treated females of NZW rabbits were inseminated with fresh doses of filtered heterospermic semen (n=27; 0.5 ml I.D. per female) and MACS separated semen (n=28; 0.5 ml I.D. per female). Separation and subsequent elimination of apoptotic spermatozoa (positive selection) from the insemination dose (after negative MACS selection) was verified under in vivo conditions on the basis of increased kindling rate in the experimental group in comparison with kindling rate in the control group (81.3% vs. 73.8%). In conclusion, elimination of apoptotic spermatozoa by the use of the MACS technique results in a slight improvement in kindling rate of rabbit does.