Influence of chemical elements on mammalian spermatozoa.

Exposure to heavy metals is the most important risk factor in the assessment of spermatogenesis. About 30-40 % cases of infertility are caused by the male factor, and most of them are due to the small quantity of spermatozoa or to inferior spermatozoa quality. The negative impact on sperm motility, morphology and concentration of such chemical elements as Al, Cr, Cd, Pb or Fe was observed, while positive influence was noticed for Zn, Mg, and Ca. The influence of Mn, Cu, Ni or Se on spermatozoa is ambiguous. Chemical elements known as necessary for capacitation and acrosome reaction are Zn, Mg and Ca, while Cd and Pb disturb initiation and progress of the acrosome reaction. The positive effect of chemical elements Al, Cd, Cr, Cu, Ni, Pb, Se, and Zn, lies in their protection against oxidative stress. On the other hand, Al, Cu and Ni induce structural changes in the testes and epididymis or influence interactions with other chemical elements.

Sex steroids level in blood plasma and ovarian follicles of the chimeric chicken.

The study was performed to determine the hormonal status of mature germline chimeras obtained by blastodermal cell transfer from chicken embryos of a donor breed [Green-legged Partridgelike breed (GP) x Araucana (AR)] to those of a recipient breed [White Leghorn (WL)] being at the same stage of embryonic development. The egg-laying chimeras and WL hens (control) of the same age were used in the experiment. At first, blood samples were taken from each bird at 0.5, 5, 12.5 and 18.5 h following oviposition. Subsequently, the chimeras and the WL hens were decapitated 1-2 h after ovulation. A stroma and the following follicles were isolated from the ovary: white normal (1-4, 4-6 and 6-8 mm), white atretic and yellow preovulatory follicles (F4-F1). Sex hormones, progesterone (P4), testosterone (T) and oestradiol (E2) in blood plasma and ovarian follicles were determined radioimmunologically. The activity of the 3beta-hydroxysteroid dehydrogenase (3beta-HSD) in the granulosa and theca layers of the follicles was analysed histochemically. In chimeric chickens, a higher level of T in blood plasma during the ovulatory cycle was noticed. However, in the stroma, white prehierarchical and medium-size preovulatory ovarian follicles the level of T was significantly lower. With respect to E2, its elevated levels were found both in blood and in the ovarian follicles. There were no significant differences in P4 concentrations in blood plasma while in ovarian follicles a higher level was observed only in white 6-8 mm follicles. 3beta-HSD activity in granulosa and theca layers of the ovarian follicles in chimeras was not different from that in the WL hens. In conclusion, the results obtained indicate that germline chimeras exhibit significant alterations in sex hormone levels in the ovary and blood plasma, which in turn may affect their reproductive abilities.

Reconstitution of a chicken breed by inter se mating of germline chimeric birds.

Blastoderm cells from chicken embryos of a donor breed (Green-legged Partridgelike; GP) were transferred to embryos of a recipient breed (White Leghorn; WL) to form chimeric progeny that, after inter se mating, permitted successful reconstitution of the donor breed. Among 23 chimeric chicks hatched from WL embryos injected with GP cells, 20 (87%) were raised until maturity, and progeny were tested by mating with GP birds to determine the ability of blastodermal cells to form germline chimeras. Six of the tested birds (30%) produced recipient-derived and donor-derived offspring, indicating that they were germline chimeras. The mean percentages of donor-derived germ cells in these birds were 21.1 (17.6 to 50.0%) and 16.9 (5.3 to 23.1%) in males and females, respectively. Among 477 chicks, resulting from mating the germline chimeric male with four germline chimeric females, 10 chicks (2.1%) exhibited a GP phenotype, indicating that the original donor stock had been reconstituted. Only one germline chimeric hen produced GP offspring, but the expected and calculated percentages of GP offspring were similar (2.99 and 2.08, respectively). Two methods of DNA analyses (RFLP and PCR amplification of polymorphic microsatellite loci) of chimeras and their offspring indicated that through mating of a relatively small number of chimeras it is possible to reconstitute a highly diverse population.

Improvement of hatchability of chicken eggs injected by blastoderm cells.

In our first experiment, we studied the effect of injection method of blastoderm cells (BC) into the subgerminal cavity of White Leghorn embryos on hatchability of chicken chimeras. Freshly laid eggs were injected through a hole made in the equatorial plane of the eggshell (Method A). In Method B, eggs were stored pointed end down for 5 to 7 d prior to injection, and a hole was cut in the blunt end of the eggshell. An advantage of Method B was that the early embryonic mortality was reduced (P < or = 0.01) and resulted in higher hatchability (41.0%; 43/105) than Method A (9.8%; 14/143). In the second experiment, we studied chicken hatchabililty as influenced by windowing (no hole, Group 1; hole in the equatorial plane, Group 2; hole in the blunt end of egg, Groups 3 and 4) and egg turning (Groups 1 and 4) or not (Groups 2 and 3) during incubation. The hatchability percentages were as follows: 67.9 (Group 1) 0.0, (Group 2) 23.3, (Group 3), and 56.8 (Group 4). A statistically significant difference (P < or = 0.05) was noted between Group 1 or 4 and the other groups. We found no statistically significant differences in the weight changes (g) but did note certain differences in the egg weight loss (%) among different egg treatments. In the third experiment, we investigated the influence of origins of BC donors: Rhode Island Red (RIR), Barred Plymouth Rock (BPR), and Green-legged Partridgelike (GP) on hatchability of putative and somatic chimera chickens. The hatchability of chimeras was dependent on the adequate assortment of BC of the donor and ranged from 7.4% (RIR) to 56.1% (GP). In the case of BC injection of the GP breed, good hatchability was accompanied by very high percentage (86.9; 20/23) of somatic chimeras.