Michael E. Kjelland

Gamete Therapeutics: Recombinant Protein Adsorption by Sperm for Increasing Fertility via Artificial Insemination

A decrease in fertility can have a negative economic impact, both locally and over a broader geographical scope, and this is especially the case with regard to the cattle industry. Therefore, much interest exists in evaluating proteins that might be able to increase the fertility of sperm. Heparin binding proteins (HBPs), specifically the fertility associated antigen (FAA) and the Type-2 tissue inhibitor of metalloproteinase (TIMP-2), act to favor the capacitation and acrosome reaction and perhaps even modulate the immune system’s response toward the sperm. The objective of this research was to determine the effect on fertility of adding recombinant FAA (rFAA) and recombinant TIMP-2 (rTIMP-2) to bovine semen before cryopreservation for use in an artificial insemination (AI) program in a tropical environment. For this experiment, 100 crossbred (Bos taurus x Bos indicus) heifers were selected based on their estrus cycle, body condition score (BCS), of 4 to 6 on a scale of 1 to 9, and adequate anatomical conformation evaluated by pelvic and genital (normal) measurements. Heifers were synchronized using estradiol benzoate (EB), Celosil® (PGF2α) (Shering-Plough) and a controlled internal drug release (CIDR) device was inserted that contained progesterone. Inseminations were performed in two groups at random, 50 animals per group. The control group was inseminated with conventional semen. The treatment group was inseminated with semen containing rFAA (25 µg/mL) and rTIMP-2 (25 µg/mL). In the control group a 16% pregnancy rate was obtained versus a 40% pregnancy rate for the HBP treatment group, resulting in a significant difference (P = 0.0037). Given the results herein, one may conclude that the HBPs can increase fertility and could be an option for cattle in tropical conditions; however, one needs to consider the environment, nutrition, and the genetic interaction affecting the final result in whatever reproductive program that is implemented.

Feathers and post-hatch eggshells: sources of fibroblast cells for conserving genetic diversity

Scientific literature addresses the use of feathers in sex determination, as a source of DNA, and for providing ecotoxicological information; however, there is much less research on the use of feathers as a non-invasive, or minimally invasive, means to conserve avian genetic diversity. This study investigated the use of semi-mature to mature feathers and post-hatch eggshells, as well as embryos, as sources of fibroblast-like cells for cytological analysis and somatic cell gene banking. Contour and flight feathers were plucked from living Rhode Island red and white leghorn varieties of the domestic chicken (Gallus domesticus), India blue peafowl (white mutation) (Pavo cristatus), domestic turkey (Meleagris gallopavo), domestic duck (Anas platyrhyncha), emu (Dromaius novaehollandiae), and ostrich (Struthio camelus). Fibroblast cell growth was observed in the feather pulp and post-hatch eggshell samples after 24-48 hours. Erythrocytes, macrophages, and epithelial cells were also observed, based on morphology. Semi-mature to mature feathers containing feather pulp and post-hatch eggshells can be adequate sources of fibroblast cells for somatic cell acquisition and cell culture, possibly providing a source of cells for use in chimera formation or cloning of threatened and endangered birds. Further research should focus on applying this technique to other avian species and address cryopreservation and viability of cells derived in this manner.

Avian cloning: adaptation of a technique for enucleation of the avian ovum

The use of reproductive technologies such as somatic cell nuclear transfer (SCNT) for avian species has been limited by the inability to visualise the pronucleus or pronuclei within the blastodisc or germinal disc region, respectively, primarily due to the opacity of the large, lipid filled yolk. The main objective in the present study was to assess a method for visualising and enucleating the avian ovum, a critical step in developing the capability for cloning birds. The method utilised in the present investigation was epi-fluorescence transmitted light (top-side UV) microscopy (EFTLM), also known as fluorescence/oblique or fluorescence/differential interference contrast (DIC) illumination, combined with vital staining and DNA visualisation techniques. The use of EFTLM combined with micromanipulation methods adapted from mammalian cloning procedures showed that the vitelline membrane of the avian ovum can be pierced and aspiration of the pronucleus, once visualised, can be performed without compromising the ovums structure. Two approaches for domestic chicken ova collection, i.e., in vivo and in vitro, were utilised and ova recovery rates were compared. Based on a statistical analysis, i.e., Fishers exact test, the results of the in vivo versus in vitro ovulation ova recovery methods were significantly different (P < 0.05), with the in vivo ovulation method yielding more viable intact ova. In conclusion, enucleating the avian ovum using EFTLM combined with vital staining, DNA visualisation, and micromanipulation techniques can be a feasible option for future avian cloning endeavours; although it will require further refinement to improve overall efficiency.

Collection, isolation, and culture of somatic cells from avian semen

Somatic cells in mammalian semen can be a potential source of nuclei for nuclear transfer to produce cloned animals. Somatic cells recovered from avian semen could also provide a source of cells for use in chimera formation or cloning of threatened and endangered birds. This type of assisted reproductive technology is especially important when a genetically unique animal has died and the only viable genetic material available is semen cryopreserved for artificial insemination and in vitro fertilization purposes. The usefulness of somatic cells obtained from fresh and frozen mammalian semen for nuclear transfer has already been evaluated, but still remains to be accomplished for avian semen. A non-invasive, or minimally invasive, technique to conserve avian genetic diversity via somatic cell collection from fresh avian semen, to our knowledge, has not been described. The present study investigated the use of fresh semen samples from domestic chickens (Gallus domesticus) (n = 7), i.e., white leghorn (n = 4) and silkie chickens (n = 3), as a source of somatic cells, specifically fibroblast-like cells and epithelial cells, for cytological analysis and somatic cell gene banking. Ultimately, somatic cell culture was successful for two of six selected samples (33.3%), with two of four white leghorn semen samples yielding cell cultures compared to 0 of 2 silkie chicken semen samples. Further research may include applying this technique to other avian species or address the effects of cryopreservation on the viability and DNA integrity of somatic cells derived in this manner.

Improvement in pregnancy outcomes in couples with immunologically male infertility undergoing prednisolone treatment and conventional in vitro fertilization preceded by sperm penetration assay: a randomized controlled trial

PurposeAnti-sperm antibodies (ASA) in men impair not only sperm motility but also fertilization and conception. However, utilization of corticosteroids to suppress ASA has shown variable pregnancy outcomes. This controversy is also extended to include the usefulness of conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) in treatments of men with ASA. This study was therefore designed to define factors contributing to these inconsistent results.MethodsInfertile men having ASA (n = 241) were randomly assigned for treatment with or without prednisolone for three cycles each of 21 days of their partner’s menstrual cycles. Control and treated men underwent then human sperm penetration assay (SPA), of hamster oocytes, to diagnose men with impaired sperm fusogenic capacity. Men with positive or negative SPA results were admitted to conventional IVF or ICSI programs, respectively.ResultsTreated patients had improved sperm motility and progressive motility when compared to control patients (P < 0.001). Fertilization (P = 0.04), embryo cleavage (P = 0.01), and chemical (P = 0.02) and clinical (P = 0.04) pregnancy rates were higher in treated patients than in control patients undergoing conventional IVF but not ICSI cycles.ConclusionsMen with ASA may also have compromised sperm fusogenic capacity, which can mask the clinical significance of corticosteroids. Corticosteroid administration in men with ASA, but without compromised sperm fusogenic capacity, improves conventional IVF but not ICSI outcomes; the reason being that ICSI bypasses issues of compromised fusogenic capacity. Inclusion of SPA in infertility clinics that offer both conventional IVF and ICSI services may be useful to identify which patients with ASA benefit from corticosteroid treatments.

Determining air cell location and embryo development in opaque shelled eggs

A novel method for determining air cell location, embryo development, and eggshell defects in opaque thick-shelled eggs was tested. Results demonstrated that a camcorder with IR “Night Shot” function performed significantly better (P < 0.05) than unaided visual determination of air cell location in Emu eggs. In addition, the Mini DVC camcorder unit was as accurate as the heavier, non-portable, and more expensive traditional IR candling units. Commercial ratite producers, waterfowl biologists, and others working with opaque thick-shelled eggs may benefit from this research.

353 EFFECTS OF CULTURE DURATION AND CUMULUS CELL REMOVAL ON CANINE OOCYTE IN VITRO MATURATION AND FERTILIZATION USING VASAL SPERM

The objective of this study was to test the hypothesis that in vitro maturation (IVM) and fertilization (IVF) rates of canine oocytes could be improved by increasing culture duration or decreasing/increasing cumulus cell contact with the oocytes when using sperm retrieved from the vas deferens. The canine oocyte is ovulated at the germinal vesicle stage, and maturation of the oocyte occurs in the oviduct and requires up to five days. Therefore, an increase in the culture duration may cause an increase in oocyte nuclear maturation. Canine ovaries and testes were collected from a local clinic, placed in warm saline solution, and transported to the laboratory. Two distinct experiments were carried out, one involving IVM (M-II) after cumulus cell removal at 72 h and 96 h for nuclear maturation evaluation, and the second experiment the same but continued up to IVF. The oocytes were recovered from the ovaries by mincing them in warm Medium-199 with Hanks salts, L-glutamine, and HEPES (GIBCO, Grand Island, NY, USA; Invitrogen Co., Carlsbad, CA, USA). Canine oocytes with a dark cytoplasm and at least 2 layers of cumulus cells were cultured in Medium-199 supplemented with Earles salts, 2200 mg mL−1 sodium bicarbonate, 25 mM HEPES, 2 mM sodium pyruvate, 5 µg mL−1 progesterone, 100 ng mL−1 epidermal growth factor, 10 IU mL−1 human chorionic gonadotropin (HCG), 5 µg mL−1 insulin, 0.50 mM epinephrine, 10p estrus bitch serum, 0.01 mM nonessential amino acids, and 20 µg mL−1 gentamicin. The oocytes were cultured for 72, 96, 120, or 144 h at 38.5°C in 5p CO2 in humidified air. The cumulus cells were removed after either a 72- or 96-h culture period. For the first 48 h, the cumulus–oocyte complexes were cultured in the modified Medium-199 containing 10 IU mL−1 HCG and then cultured in the same medium free of HCG. The oocytes were denuded by pipetting, stained with Hoechst 33342, and examined for nuclear maturation. ANOVA was used for statistical analysis of the data. The IVM rate (MII) was significantly higher (P 0.05). The sperm motility index (SMI e motility percentage × sperm activity grade) was significantly higher in sperm retrieved from the vas deferens (vasal sperm) compared to epididymal and testicular sperm (259 vs. 95 and 19.2, respectively, P 0.05). Sperm penetration was significantly higher at 96 h compared to 72 h, and the number of sperm heads inside the ooplasm was 3.2 for the 72 h group vs. 4.8 for the 96 h group (P < 0.05). In conclusion, increasing the IVM culture period beyond 72 h did not increase the oocyte maturation rates, and increasing the culture time to 96 h without cumulus cells present increased the rate of sperm penetration.