Testis Size, Sperm Quality, and Seminal Fluid Parameters of Gray (Canis lupus) and Mexican Gray Wolves (C.l. baileyi)

Introduction - Although sperm and seminal fluid of domestic dogs has been extensively studied, typically as part of artificial insemination programs, limited comparative data exist for other canid species. Following the request in 1990 for the establishment of a sperm cryopreservation bank for the highly endangered Mexican gray wolf (Canis lupus baileyi), we began collecting semen samples during the annual breeding season. In 2000, we began using gray wolves (C. lupus) as a model for the Mexican gray wolf subspecies in developing and testing aspects of assisted reproduction. We have since evaluated semen samples from 61 Mexican and 18 gray wolf males, many of them in multiple years and some multiple times in one year.

Materials and Methods - Samples were collected under anesthesia using electroejaculation (see Asa et al. 2007 for methodology). Standard semen analysis included percentage of motile sperm, status, sperm concentration, percentage of morphologically normal cells, pH and osmolality of seminal fluid. Motility was assessed using phase-contrast microscopy at 200X; morphology was assessed at 400X or 1000X following either eosin-nigrosin or Spermac staining. Osmolality of seminal fluid was measured with an osmometer after centrifugation to remove sperm, and pH of semen was quantified either by paper strips or a pH meter. In addition, we measured individual testes (length plus two measures of width for each) using calipers.

Results - In a previously published study (Asa et al. 2007) we correlated percentages of motile and of morphologically normal sperm with measures of inbreeding in Mexican wolves, but other aspects of semen quality were not reported. Here we summarize our years of results for additional parameters with more detail for sperm morphology and motility.

All samples were collected during the breeding season, from late January through early March. All males were adult, i.e., 22 months of age or older, except one Mexican wolf that reached puberty at 10 months, documented by the presence of good quality sperm. Although males typically reach puberty at 22 months, this male had been hand-raised during a critical socialization period, then returned to its natal group, perhaps disturbing natural kin- recognition processes. At 10 months it began to compete with its father and court its mother, something that would not occur in a properly functioning family group.

Testis volume was approximated by multiplying the three measurements, since even the formula for an ellipsoid does not truly represent the shape of the typical wolf testis. Although the simple product of the three measures is not an accurate volume, it can provide a relative comparison between the species and subspecies. Unfortunately, no testis measures are available for the non-breeding season. Mean volumes for gray wolves were 38.8±1.4cm3(left) and 36.8±1.6cm3 (right) and for Mexican wolves 33.9±0.5 and 35.1±0.5cm3 for left and right sides, respectively. Gray wolf testes may be slightly larger because of their larger body size. For our study animals, body weights ranged from about 40-55kg for the male gray wolves and 30-35kg for the Mexican subspecies.

Measures of seminal fluid pH and osmolality were similar for both groups. Mean pH (±SE) was 6.7±0.01 and 6.9±0.01 for gray and Mexican wolf semen, respectively. Osmolality was 332.4±2.1 and 332.3±0.9 for gray and Mexican wolf seminal fluid, respectively.

Considered overall, Mexican wolves had the poorest sperm samples, with some individuals having 100% abnormal forms, whereas the poorest value for gray wolves was 45% abnormal (i.e., 55% normal). In contrast, the best gray wolf samples had 96% normal and best Mexican wolf 95% normal. For gray wolves, the majority of abnormalities were distal cytoplasmic droplets, abnormal acrosomes, and kinked tails. For the Mexican wolves, there were many more types of abnormalities, including microcephalic or detached heads, proximal or distal cytoplasmic droplets, coiled or kinked midpieces, and kinked or broken tails. As reported in Asa et al. (2007) sperm quality, as measured both by percentages of motile and morphologically normal sperm, was generally higher in gray wolves. However, when highly inbred Mexican wolf males were removed from the analysis, the differences disappeared.