MOLECULAR REPRODUCTION A N D DEVELOPMENT 37:457461(1994)

Spermophagy in Semen in the Red Wolf, Canis rufis JAMES K. KOEHLER,' CARROL C. PLATZ, JR.,' WILL WADDELL: MICHAEL H. JONES: ROLAND SMITH: AND SUSAN BEHRNS3 'University of Washington, School of Medicine, Seattle, Washington; 21nternational Canine Semen Bank, Sandy, Oregon; and 3Point Defiance Zoo and Aquarium, Tacoma, Washington

ABSTRACT The red wolf (Canis rufus) is an en- dangered species with 194 individuals remaining in the wild and in various captive facilities. Breeding efforts at the Graham, WA site (Point Defiance Zoo and Aquarium) have involved artificial insemination with fresh or frozen semen in an effort to increase population and maximize the ge- netic potential of the stock. Electron microscopic observa- tions were made in semen specimens obtained by electro- ejaculation from mature males prior to their use in an effort to determine semen parameters that might be useful in guiding breeding procedures. Sperm samples were either fixed immediately or treated with capacitating media and fixed after 4 to 7 hr of incubation. Many of the specimens examined were pyospermic (white cell in semen) and showed evidence of spermophagy, primarily by neutro- phils. Of the six animals surveyed, only one showed little evidence of spermophagy, and three had extensive py- ospermia and spermophagy but this finding was not corre- lated with fertility. Samples fixed immediately as well as those incubated for several hours showed evidence of spermophagy, indicating that the phagocytosis was not the result of culture. Gene pool restriction and/or captive stress may be contributing factors of reduced semen qual- ity. 0 1994 Wiley-Liss, Inc.

Key Words: Canine sperm, Pyospermia, Ultrastructure

INTRODUCTION Taxonomists generally agree that the living, wild

canid species in North America include the coyote (C. latrans), the gray wolf (C. lupus), and the red wolf (C. rufus). Whereas the gray wolf has an extensive range in North America, the red wolf was largely restricted to the Southeastern United States extending westward to Texas and Northern Mexico. Human intervention ulti- mately created a set of circumstances that simulta- neously devastated red wolf habitat and populations. The red wolf was designated an endangered species in 1967 and was declared extinct in the wild by 1980. As of 1990, only 130 animals remained in existence, almost all of them in various captive and breeding facilities (US Fish and Wildlife Service, 1989). Currently, 194 animals are accounted for in the wild and in captivity. A lively and extended debate on the species designation of the red wolf has centered on a postulated hybrid (coyotelwolf) origin for the animal as contrasted to a more primitive line, retaining characteristics of an an- 0 1994 WILEY-LISS, INC.

cestral stock from which both wolves and coyotes arose (Nowak, 1979, 1992). Recent molecular data on mito- chondrial DNA (Wayne and Jenks, 1991) has rekindled the debate concerning the species or hybrid status of the red wolf (Wayne, 1992; Phillips and Henry, 1992). The US Fish and Wildlife Service position is that the present data "do not provide substantial support for the contention that the red wolf is a wolflcoyote hybrid" (Henry, 1992). Efforts to enhance the population of this endangered species and to study and maximize its ge- netic potential have led to an artificial insemination program and associated analyses of semen quality and other reproductive parameters. This effort to promote artificial modes of reproduction has been initiated at the red wolf program's off-site breeding facility in Gra- ham, WA. Electron microscopy was used to monitor the semen quality of electroejaculated specimens before and after incubation in capacitation media. This report describes electron microscopic observations made on red wolf semen samples obtained during the 1992 breeding season at this facility.

MATERIALS AND METHODS Animals and Semen Aquisition

The red wolf population used is located at the Captive Breeding Facility of The Point Defiance Zoo, Graham, WA. Animals are housed in large open pens with natu- ral foliage and concrete dens. Males a t the site were sampled approximately every 2 weeks during the breeding season, unless ill, a frequency felt to minimize anesthesia trauma. Samples used in this study were collected during the spring 1992 breeding season, spe- cifically on 2/26,3/6,3/18, and 413. Wolf #224 was used on all four dates, wolf #369 on two, and all others just once. For semen collection, wolves were captured ac- cording to techniques established by the Red Wolf Spe- cies Survival Plan, transferred to holding cages, trans- ported a short distance to an indoor collection site, and anesthetized with 175 mg Telazol (tiletamine HC1 and zolazepam HC1) i.m. Electroejaculation, using a P-T Electronics Model 302 and #4 (1.6 cm dia) rectal probe, was carried out according to the procedure of Platz and

Received May 1,1993; accepted September 13,1993. Address reprint requests to James K. Koehler, Ph.D., Biological struc- ture, SM-20, University of Washington, School of Medicine, Seattle, WA 98195.

458 J.K. KOEHLER ET AL.

TABLE 1. Status of White Blood Cell Presence and Spermophagy in Semen

Birth Wolf#" date

Demonstrated Sire#/Dam# Fertilityb

224 4/23/83 247 4/19/84 268 4/23/85 312 4/26/87 36gd 5/3/89 490 5/10/91

- 11/54 11/54 + 53/79 +

2421279 -

2911233 + 2721195 +

White cells' Spermophagy Litter datesfnotes

+ + 3 A.I. attemptse

+ + + 3 A.I. attempts

++ ++ 1987,1992 1988,1989, 1992 -

++ ++ 1992 ++ ++ 1993

"Wolf identification numbers are cataloged in Parker et al. (1990). b+: Proven fertile; -: not proven fertile.

dry field and extensive spermophagy. dWolf # 369 is unilteral cryptorchid or monorchid. "A.I.: Artificial insemination.

+: Greater than one white cell per high dry (400 X ) field and spermophagy observed. + + : Greater than 10 white cells per high

Fig. 1. Red wolf sperm fixed immediately after ejaculation showing typical morphology of canine spermatozoa including fully condensed nuclear chromatin (Nu), tightly adherent acrosome (Ad, and postac- rosomal region (PAC). Plasma membrane overlying the acrosome is artifactually ruffled. 23,600x.

Fig. 2. Red wolf sperm maintained in HBS for 3 hr prior to fixation showing that no detectable morphological alterations have occurred

during this time period (compare with Fig. 1). Sloughed epithelial cells (*) are commonly seen in the ejaculates. 21,500 x .

Fig. 3. Incubation for 6 hr in CCM (see text) triggers the acrosome reaction in some of the sperm as seen in this figure. Mosaic membrane vesicles (arrows) are seen surrounding the evacuated acrosome. An unreacted sperm (to the left) is also present. 26,400~.

Seager (1978). Three stimulation periods were used for collection with 5 min rest intervals. Multiple stimula- tions (25-30) were used during collection with semen

collected in small plastic beakers. No attempt was made to fractionate the ejaculates. Semen was pooled and evaluated for motility and concentration after cen-

RED WOLF SEMEN 459

sperm phagocytosis, although even in this case (#268) some sperm were seen to be attached to white cells.

The extent of white cell infiltration and sperm phago- cytosis in some of these samples can be appreciated by examining Figure 4, a light micrograph showing a clus- ter of phagocytic cells, some of which contain sperm. Few free sperm were seen in these preparations. Figures 5 and 6 illustrate the electron microscopic ap- pearance of phagocytic white cells in a number of se- men samples examined in this study. Spermophagy was seen in samples fixed immediately (Fig. 5) as well as in samples incubated with capacitation media (Fig. 6), indicating that such activity was not a result of postejaculation culture. Nuclear and granule morphol- ogy of these cells suggests they were of neutrophilic origin, but mononuclear phagocytes were occasionally also seen. Many of the attached and incorporated sper- matozoa did not appear to be morphologically defective or abnormal, suggesting that the phagocytosis was gen- eralized rather than targeted. In some cases, a special- ized membrane complex appeared to develop between the phagosome membrane and the sperm plasma mem- brane (Fig. 7). A uniform, dense layer was observed between the membranes and, in these cases, there ap- peared to be little or no morphological degradation of the spermatozoa.

DISCUSSION The semen quality of this sampling of red wolves was

dramatically impacted by white cell infiltration (py- ospermia) and spermophagy. Sperm phagocytosis has been well documented in human and other primates following vasectomy (Alexander, 1975) and in cases of infective and idiopathic prostatitis (Berger et al. 1979; Koehler, 1983), but has not been extensively studied in other animals. Although the penis of the animals is carefully wiped prior to semen collection, it is possible that some white cells are picked up from the remnants of smegma which collects in the foreskins of some of the males.

Four of the animals of this group are of proven fertil- ity (Table 1). One of these animals (268) showed some pyospermia, but no phagocytosis of sperm, whereas the other three (#247,369,490) showed extensive pyosper- mia and phagocytosis. In the human, pyospermiat spermiophagy has been shown to be a negative indica- tor of fertility (Berger et al. 1982), thus it was somewhat surprising to find that three of four fertile animals showed significant pyospermia and spermoph- agy. Observations on this small sample of animals sug- gests no correlation of fertility and spermophagy in the red wolf, but more data on larger populations is needed before any generalizations can be made. Interestingly, work with the cheetah (Wildt et al., 1983) suggests that the finding of a very high proportion of abnormal sperm (>50%) in this endangered species may be due to ge- netic effects of inbreeding. Although not directly com- parable to the findings here, the present abnormalities may be the result of a decreasing gene pool available to the red wolf population. Captive stress, as well as

trifugal washing in phosphate-buffered saline (PBS). Three media were qualitatively evaluated with regard to sperm incubation prior to fixation in addition to an aliquot fixed immediately without incubation. These included Hanks balanced salt solution (HBS), a capaci- tation medium described by Mahi and Yanagimachi (1978) for domestic canines (CCM), and the culture me- dium (BWW) of Biggers et al. (1971), as modified by Koehler et al. (1982). Sperm remained in these media in a water bath at 35°C at ambient atmosphere for periods of 4-7 hr and were processed for electron mi- croscopy to determine the status of the acrosome and other fine structural parameters. An estimation of white cells in the semen was made by examining a 10-fold dilution of the fixed cells a t 4 0 0 ~ in phase con- trast and categorizing them as less than one cell, greater than one cell, or greater than 10 cells per field (Table 1).

Electron Microscopy Small aliquots of the above preparations were fixed

for electron microscopy in 1.25% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3, at room temperature for a t least 1 hr. Specimens were transported to the labora- tory for further processing including postosmication in 1% OsO, (cacodylate buffer), ethanol, and propylene oxide dehydration and embedding in Epon 812. Thin sections were prepared with a Sorvall PB-I1 ultramicro- tome equipped with a diamond knife, stained with ura- nyl acetate and lead citrate, and examined in a Philips 420 transmission electron microscope.

RESULTS Light microscopic examination indicated that the

HBS solution was capable of maintaining sperm in a stable, motile condition for several hours after collec- tion. Figure 1 shows a red wolf sperm fixed immedi- ately after collection demonstrating the typical ultra- structural appearance of canine spermatozoa with fully condensed chromatin, intact acrosome, and tightly ad- herent postacrosomal sheath. The plasma membrane overlying the acrosome shows the typical (probably ar- tifactual) tendency to ruffle from the head surface. Figure 2 shows a similar image of a sperm incubated for 3 hr in HBS. Also present are portions of epithelial cells, presumably sloughed during ejaculation, which are frequently seen in these seminal samples. It was further observed that the capacitation medium of Mahi and Yanagimachi (1978), CCM, was clearly superior to BWW in producing an acrosome reaction in a signifi- cant fraction of sperm over a 7 hr period (Fig. 3). All media tested resulted in a decreasing motility of the sperm over a 5-6 hr period.

Table 1 identifies the animals used for the electron microscopic observations, their age, and the presence of white blood cells and/or phagocytosis of spermatozoa as detected ultrastructurally. The results indicate that all of the animals showed significant numbers of seminal white cells. Three of the animals had extensive sper- mophagy and only one of the six had no demonstrable

460 J.K. KOEHLER ET AL.

Figs. 4-7.

RED WOLF SEMEN 461

trauma associated with electroejaculation, could also result in abnormalities in seminal characteristics, but it is difficult to conceive how white cell infiltration and spermophagy could be a common denominator for such stress-induced trauma.

These studies will be pursued with a larger sample of animals to determine if the seminal characteristics here reported, particularly the spermophagy, are wide- spread and of long-term duration. If confirmed it may be possible to improve semen quality with antibiotic or drug therapy.

ACKNOWLEDGMENTS This research was partially supported by a grant to

J.K.K. from the Center for Wildlife Conservation. The authors express their thanks to W. Dianne Smith, Re- productive Assays Laboratory, Tacoma, WA, for ini- tially suggesting this study, to Dale Peterson for his continued work in the maintenance of the red wolf pop- ulation at the Graham facility, and to Ms. Tracy Cran- ick for word processing expertise.

Fig. 4. Light micrograph (1 micron, toluidine blue, epoxy section) showing cluster of phagocytic white blood cells in red wolf semen. A number of sperm have been incorporated into the cells (arrows), most of which appear to be neutrophils. Note the virtual absence of free sperm in this field. 2,200X.

Fig. 5. Electron micrograph of spermophage in red wolf semen con- taining endosomes with engulfed sperm (sp) and midpiece (M); multi- lobulated nucleus (Nu) and several surface-attached sperm (arrows) are also visible. 12 ,000~ .

Fig. 6. Spermophage in semen sample incubated 4 hr in CCM incor- porating endosomes with sperm nuclei and flagellar components. Cell nuclei (Nu) also shown. 16,700x.

Fig. 7. Sperm enclosed in an endosomal vesicle of a spermophage in red wolf semen fixed immediately after collection. Note the dense membrane complex (arrows) surrounding the sperm head. 18,500 x.

REFERENCES Alexander NJ (1975): Immunologic and morphologic effects of vasec-

tomy in the rhesus monkey. Fed Proc 34:1692-1697. Berger RE, Alexander ER, Harmisch JP , Paulson CA, Monda GD,

Ansell J , Holmes KK (1979): Etiology, manifestations and therapy of acute epididymitis: Prospective study of 50 cases. J Urol 121:750- 754.

Berger RE, Karp LE, Williamson RA, Koehler JK, Moore DE, Holmes KK (1982): The relationship of pyospermia and seminal fluid bacte- riology to sperm function as reflected in the sperm penetration assay. Fertil Steril37557-564.

Biggers JD, Whitten WK, Whittingham DG (1971): The culture of mouse embryos in vitro. In J C Daniel, Jr. (ed): “Methods in Mamma- lian Embryology.” San Francisco: W.H. Freeman and Co, pp 86116.

Henry VG (1992): Endangered and threatened wildlife and plants: Finding on a petition to delist the red wolf (Canis rufus). Federal Register 57:12461250.

Koehler JK, DeCurtis I, Stenchever MA, Smith D (1982): Interaction of human sperm with zona free hamster eggs: A freeze fracture study. Gamete Res 6:371-386.

Koehler JK, Berger RE, Smith D, Karp LE (1983): Spermophagy. In ESE Hafez and P Kenemans (eds): Atlas of Human Reproduction.” Boston: MTP Press, Chapter 22, pp 213-217.

Mahi CA, Yanagimachi R (1978) Capacitation, acrosome reaction and egg penetration by canine spermatozoa in a simple defined medium. Gamete Res 1:lOl-109.

Nowak RM (1979) North American quarternary canis. Museum of Natural History, Univ. of Kansas Monograph No. 6, Lawrence, KS.

Nowak RM (1992) The red wolf is not a hybrid. Conserv Biol 6593- 595.

Phillips MK, Henry VG (1992) Comments on red wolf taxonomy. Con- serv Biol 6:596599.

Platz CC, Seager SWJ (1978): Semen collection by electroejaculation in the domestic cat. J Am Vet Med Assoc 173:1353-1355.

U S . Fish and Wildlife Service (1989) Red Wolf Recovery Plan. At- lanta, GA. U.S. Fish and Wildlife Service.

Wayne RK (1992) On the use of morphologic and molecular genetic characters to investigate species status. Conserv Biol6:590-592.

Wayne RK, Jenks SM (1991): Mitochondria1 DNA analysis implying extensive hybridization of the endangered red wolf (Canis rufus). Nature 351:565-568.

Wildt DE, Bush M, Howard JG, OBrien SJ, Meltzer D, Van Dyk A, Ebedes H, Brand DJ (1983): Unique seminal quality in the South African cheetah and a comparative evaluation in the domestic cat. Biol Reprod 29:1019-1025.