Breeding soundness refers to a male's ability to get females pregnant. Therefore, the breeding soundness examination (BSE) involves a complete and systematic evaluation of the reproductive potential of a given male. No single measurement or criterion reliably predicts fertility, and therefore several criteria are usually evaluated, including breeding ability and libido, general physical examination and inspection of the genital organs, as well as assessment of sperm production and quality.
The BSE is not a direct evaluation of fertility: this can be confirmed only by successful production of offspring after the male breeds a proven fertile female. The specific male animal must be properly identified, and a detailed history is important because sub- or infertile males might require more exhaustive evaluation. The evaluation of breeding ability and libido is only partially possible when collecting semen via artificial vagina or manual stimulation in the presence of a female in estrus. Therefore, breeding ability is seldom evaluated in traditional routine BSEs for bulls and rams in which semen is typically collected via electroejaculation.
The basic components of semen quality evaluation are the following:
gross semen appearance
semen volume and sperm concentration, which allow for the calculation of the total number of sperm in the ejaculate
sperm motility, including gross motility (ruminants only) and percent individual sperm motility (total and progressive) of a raw and/or diluted sample
the percent morphologically normal sperm
A Romanowsky-stained cytology sample also allows for evaluation of red (hemospermia) or white (pyospermia) blood cells in the ejaculate. Additional sperm viability or sperm function tests can be performed in semen freezing centers or laboratory settings (see Ancillary Sperm Tests below). When collecting semen from ruminants via electroejaculation, the sperm production potential is estimated by measuring scrotal circumference. Scrotal circumference is correlated with daily sperm output and therefore the serving capacity of a bull or ram (ie, number of females it can impregnate in a limited time).
After the BSE is complete, the male is classified as a satisfactory, questionable, or unsatisfactory prospective breeder. An animal with physical defects that may be inherited (including cryptorchidism) should be declared unsatisfactory. Guidelines for the BSE in each domestic species are included in the species-specific discussions of bulls, rams, bucks, and male dogs.
Also see Management of Reproduction: Pigs.
Ancillary Sperm Tests
Ancillary sperm tests are tests that aim at evaluating membrane integrity and/or some aspect of sperm function. Because sperm plasma membrane integrity is essential for sperm viability and function, this should be evaluated when feasible. Eosin is a stain that penetrates disrupted membranes; stained sperm that appear pink to red under light microscopy are not viable. Several fluorescent probes can also be used; however, they require a laboratory equipped with a fluorescent microscope and hence their use is restricted to referral laboratories or some semen freezing centers. In addition, fluorescent probes can be used in combination with fluorescent-activated cell sorting (FACS), allowing for fast evaluation of thousands of spermatozoa. Fluorescent probes can be thus combined to provide a reliable assessment of membrane integrity or sperm function. An example is SYBR-14 and propidium iodide, which label viable (green) and non-viable (red) sperm, respectively.
The integrity of the sperm DNA is also essential for normal embryo development after fertilization. In this regard, the sperm cell structure assay (SCSA) measures the ability of sperm chromatin to withstand acid denaturation using the probe acridine orange and FACS. Acridine orange will emit green or red fluorescence when binding to double (intact) or single (denatured) stranded DNA, respectively. In essence, properly packaged, nucleosome-bound sperm DNA should be able to withstand acid denaturation and would be detected green under fluorescence microscopy or FACS.
Some tests provide a more direct evaluation of sperm function and involve assessing some aspect of the fertilization process. Examples of such tests include the evaluation of sperm capacitation parameters, ability to undergo acrosomal exocytosis, sperm-zona pellucida binding, sperm-oocyte penetration, and/or pronuclear formation after in vitro fertilization (IVF). Notably, these tests are time consuming and require high expertise and specialized facilities; they are seldom performed in routine BSEs or the clinical situation. In addition, many of these ancillary tests have not been validated for all domestic species.
Spermatogonial Stem Cells and Prospects for Preservation of Fertility in Male Animals
Spermatogonial stem cells (SSCs) are stem cells located against the basement membrane of the seminiferous tubules. As such, they can regenerate themselves and also give rise to daughter cells that follow the replication and differentiation path toward the generation of mature sperm. Of particular interest is that SSCs are present throughout the life of a given male and hence may provide an avenue to preserve or even restore male fertility.
Although much work is still needed for SSCs to become a tool in the management of male infertility, these cells have been isolated and partially characterized in boars, bulls, tom cats, and stallions. Most knowledge derives from studies in the mouse, in which SSCs can be cultured indefinitely, cryopreserved, genetically modified, and transplanted into the testes of sterile recipient mice for generation of mature sperm cells. Studies are underway to pursue these goals in domestic species.
In the future, SSCs may be used to preserve fertility in valuable domestic species as well as in their endangered wild counterparts (eg, felids). Moreover, SSCs could also become an integral tool in the assessment of male fertility or ability to restore fertility in a given male. In addition, and given some of the advances in assisted reproductive technologies in domestic species, it is feasible that SSCs could be differentiated into haploid germ cells in vitro for intracytoplasmic sperm injection and the generation of offspring from valuable azoospermic males.
