Abstract
The occurrence of a high incidence of sperm tail defects in a male domestic cat resembling the known ‘Dag-like’ defect is reported. Sperm analyses were performed in ejaculated samples collected by an artificial vagina and in testicular and epididymal sperm cells after castration. The following alterations were observed using transmission electron microscope: heavily coiled sperm tails containing several axonemal units enclosed in the same common cell membrane; aberrations in the axonemal main structure; and swollen and unevenly distributed mitochondria in the midpiece. Abnormal modifications in the mitochondrial sheath were also found in sperm cells retrieved from testes and epididymides. Considering these findings, we can conclude that this is the Dag-like defect, described previously in other domestic species and a testicular origin may be involved.
The ‘Dag’ or ‘Dag-like’ defect is a sperm tail defect characterized by multiple fractures of the axonemal fibers, and mitochondrial sheath disruption. This defect was first described in a Jersey bull named Dag. 1 Since then, this sperm abnormality has been reported the equine, 2 caprine 3 and canine species. 4 According to Barth and Oko, 5 the Dag-like defect is found commonly in various bull breeds, affecting <5% of the spermatozoa from an ejaculate, and can lead to subfertility when incidence exceeds 50%.1,6 Based on a breeding experiment, Koefoed-Johnsen et al 7 confirmed that the Dag defect in the Jersey breed is due to an inherited recessive factor.
A high incidence of morphologically abnormal sperm is commonly found in the domestic cat and when >60% of spermatozoa per ejaculate are affected, the animal is termed teratospermic. 8 Various sperm defects have been found in the cat, comprising head, acrosome and tail abnormalities, detached heads and proximal and distal cytoplasmic droplets; even a low incidence of the Dag-like defect may have been observed.8–11 To date, no report based on the ultrastructural analysis of the Dag-like defect is available in the domestic cat. Therefore, this report aims to describe a case of a teratospermic cat showing a high incidence of the Dag-like defect, using ultrastructure evaluation. Additionally, testosterone, selenium and zinc concentrations, and sperm quality were also assessed. Results were compared with normospermic males.
Material and methods
Animal profile
A mixed-breed long-haired male cat aged 5 years, donated by its owners for personal reasons, was housed in the veterinary school research cattery for approximately 2 years. During this period, the tom cat was used as a teratospermic semen donor for research purposes, as it continuously presented a high incidence of abnormal sperm morphology, especially tail defects. Afterwards, the animal was neutered and adopted by a new family.
In the research cattery the cat was exposed to natural lighting (>12 h of light/day; 22°53’09’’S; 48°26’42’’W), and provided with dry cat food (FIT 32; Royal Canin) and water ad libitum. The male had visual and olfactory contact with intact male and female cats, and direct contact with a trained non-estrus queen during the semen collection procedures. Unfortunately, no reproductive history of the animal was available before it arrived at the research cattery. Physical examination of the tom cat revealed neither alterations in health condition nor abnormalities of the genital system. In addition, the male presented normal libido and was serum negative for both feline immunodeficiency virus and feline leukemia virus.
In order to compare testosterone, selenium and zinc concentrations, sperm quality and testicular volume, three normospermic cats (more than 60% morphologically normal sperm) aged 4–7 years were used. Collection of blood and semen samples and testicular measurements from these males were performed using similar methodology, frequency and period as described below for the teratospermic cat. Likewise, all normospermic cats were housed in the research cattery and, hence, submitted to equal lighting, social and nutritional conditions as the tom cat of this case report.
Testicular volume, and testosterone, selenium and zinc measurements
For all tom cats, testicular measurements were made using a laboratory caliper (length and width). The total testicular volume was calculated as the sum of the right and left testis volume using a formula described previously. 8 The total testicular volume obtained for the teratospermic cat (3.61 cm3) was slightly higher than the 95% confidence interval (CI) calculated with the results from the normospermic cats (2.21–3.50 cm3).
Blood samples from all cats were collected under manual restraint on two different occasions (November and December) and always at the same hour of the day. Total serum testosterone concentration (T level) was measured by a commercial solid-phase radioimmunoassay kit (Coat-A-Count kit; Diagnostics Products Corporation) and the intra-assay coefficient of variation was 4.8%. Interestingly, the cat presenting the Dag-like defect showed a mean T level (178.0 ± 77.9 ng/dl) within the 95% CI obtained from the normospermic animals (115.6 to 240.3 ng/dl).
Sperm samples (10–15 ejaculates/cat) were collected from October to December, with at least a 2-day interval between collections and using an artificial vagina and a non-estrus queen or inanimate object for teasing. These samples were used to assess seminal plasma zinc (SPZn) and selenium (SPSe) concentrations. To achieve this goal, seminal plasma was separated from sperm cells using two consecutive centrifugations (5 mins at 700 × g and 15 mins at 1500 × g) and stored at −80°C. Before SPSe and SPZn determination, samples from the same tom cat were thawed, pooled and centrifuged at 3000 × g for 10 mins. Blood selenium (BSe) and zinc (BZn) concentrations were assessed using the same pooled serum samples collected for T level determination. Selenium and zinc concentrations were measured by a Shimadzu AA-6800 atomic absorption spectrometer using the methods described by Neves et al 12 and Silva et al. 13 Among the values obtained for selenium and zinc only SPSe for the teratospermic cat (42.0 µg/l) was lower than the 95% CI calculated for the normospermic animals (53.0 to 54.4 µg/l).
Semen evaluation
For sperm analyses, each cat was collected three times with a 2-day interval between collections, and using an artificial vagina as described above (November). After dilution in Talp HEPES 6% bovine serum albumin, total sperm count was determined and sperm motion assessed by computer assisted sperm analysis (HTM – IVOS 12; Hamilton Thorne Research) set up previously for cats. 14 Plasma membrane integrity was evaluated with propidium iodide (5 µg/ml; Sigma Chemical) and carboxyfluorescein diacetate (9.2 µg/ml; Sigma Chemical), 14 while sperm nuclear chromatin normality was assessed using acridine orange stain (10 mg/ml; Sigma Chemical) by the method modified for cats. 15 A total of 200 cells per sample were analyzed in each test using an epifluorescence microscope [Nikon, Episcopic Fluorescence Attachment (EFA) Halogen Lamp Set]. Additionally, sperm samples were fixed in formol–saline for morphological evaluation and also stained with fast green FCF and rose bengal to assess acrosome integrity. 16 For both methods, 200 cells per sample were counted using light microscope (1000 × magnification). Morphology of sperm cells retrieved from testes (rete testis and efferent ducts) and epididymides after castration of the teratospermic cat was also assessed in wet preparations with formol–saline.
Data for sperm quality are shown in Table 1. The values of sperm motility, sperm velocity, rapid sperm percentage and DNA integrity found for the cat showing the Dag-like defect were lower when compared with the 95% CI calculated for the normospermic cats.
Mean ± standard deviation values of sperm quality for the normospermic cats (n = 3) and the teratospermic cat (n = 1) showing the Dag-like defect assessed from nine and three total ejaculates, respectively
Value lies outside the 95% confidence interval established for normospermic cats
ALH = amplitude of lateral head displacement; BCF = beat cross frequency; Rapid = VAP >medium VAP cut-off; VAP = average path velocity; VCL = curvilinear velocity; VSL = straight line velocity
The most common morphological defects found in the ejaculated sperm from the teratospermic cat were 32.3 ± 1.7% of bent tail with or without cytoplasmatic droplet, 26.8 ± 4.5% of Dag-like defect (strongly coiled, folded or fractured midpiece) (Figure 1), 12.5 ± 2.8% of macrocephalia, 11.5 ± 3.4% of abnormal acrosome and 4.8 ± 1.2% of distal cytoplasmic droplet. Furthermore, among the sperm cells displaying the Dag-like defect, 38.1% of them presented a macrocephalic head (Figure 1). Sperm cells from testes and epididymides also presented a high percentage of macrocephalic spermatozoa (Figure 2). Interestingly, epididymal sperm cells exhibited a higher incidence of the Dag-like defect when compared with the cells retrieved from testes (Figure 2). Likewise, the percentage of sperm cells affected simultaneously by both the Dag-like and the macrocephalic defects also increased in the epididymides.
Phase contrast micrograph of wet smears from formol–saline-fixed spermatozoa of ejaculate origin. (a) Tightly coiled tail and macrocephalia. (b) Fractured midpiece and macrocephalia. (c) Bent tail with normal size head (arrow) and tightly-coiled-tail spermatozoa showing macrocephalic head (arrowhead) (× 1000)
Percentage of testicular, epididymal and ejaculated sperm cells showing the Dag-like defect, macrocephalia, and both the Dag-like defect and macrocephalia simultaneously
Testicular, epididymal and ejaculated spermatozoa from the cat showing a high incidence of the Dag-like defect were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3) and then prepared for ultrastructural analysis as described previously. 17 In brief, the material was post-fixed in 1% osmium tetroxide solution for 2 h, dehydrated through a graded series of acetone and embedded in epoxy resin. Ultra-thin sections were double-stained with uranyl acetate and lead citrate, then examined under a Philips C100 transmission electron microscope (TEM).
The ultrastructural investigation of the Dag-like defect revealed a flagellar compartment consisting of strong folding, coiling and fracture of the midpiece. Furthermore, coiled tails were encapsulated in a thin membrane, which trapped several membranous organelles, as found in the cytoplasmic droplet (Figure 3). Midpieces presented disarranged mitochondrial sheaths with irregularly sized, swollen and unevenly distributed mitochondria (Figures 3 and 4). Transverse sections of the tail revealed changes in the axonemal structure, such as lack of one or more doublets microtubules, often associated with the absence or displacement of the corresponding outer dense fiber. Doublets loss was usually found affecting the half of the axoneme containing fibers 4, 5, 6 and 7 and sometimes even the central microtubule pair (Figures 4 and 5). Microtubules were occasionally observed outside the fibrous sheath, loosely found in the residual cytoplasm (Figure 4).
Longitudinal section of a sperm cell from ejaculate samples showing coiled tail encapsulated by a common membrane, and swollen and unevenly distributed mitochondria in the midpiece (transmission electron microscope; bar = 1 µm)
Longitudinal section of ejaculated spermatozoa displaying cross sections of abnormal axonemes (A–C). In sections A and B the axoneme lacks doublets microtubules 4, 5 and 7, and 4, 5 and 6, respectively. The axonemal unit in C shows a dislocated central microtubule pair (arrowhead) and some of the doublets are missing. Doublets microtubules are found outside the axoneme, but within the plasma membrane (arrow) (transmission electron microscope; bar = 0.2 µm)
Cross sections of ejaculated sperm cells’ midpiece. In section A doublets microtubules 6 and 4, with the corresponding outer dense fiber, are missing. In B the axonemal unit lacks four doublets microtubules and the central microtubule pair (transmission electron microscope; bar = 0.5 µm)
Ultrastructural analysis of the epididymal spermatozoa showed similar morphological alterations as described earlier for the ejaculated samples. Furthermore, although the majority of the cells from rete testis and efferent ducts exhibited a normal-appearing axial fiber bundle, some cells presented a swollen and misaligned mitochondrial sheath and the accumulation of a granular material in the residual cytoplasm.
Discussion
According to Andersen Berg et al, 18 breed-related differences between Dag and Dag-like defects exist in the bovine species. While the latter defect is characterized by the presence of mitochondria misalignment and malformation of the axial fiber bundle, these structural alterations were not reported in the Jersey bulls affected by the Dag defect.1,19 Whether these two sperm defects constitute separate entities remains a question to be answered. Based on the ultrastructural changes found in the sperm cells from the cat of this case report, we can confirm the occurrence of the Dag-like defect in domestic cats, as reported previously in other species.2–5,18 Moreover, this cat also exhibited a high incidence of other sperm abnormalities, such as bent tail with or without cytoplasmic droplet and macrocephalia, which are common findings in teratospermic cats.8,20
Regarding the sperm motion parameters, as expected, the high incidence of sperm tail defects displayed by the teratospermic cat clearly impaired sperm motility and velocity. In addition, the teratospermic cat from this report showed lower DNA integrity when compared with its normospermic counterparts, which is in accordance with the findings of Penfold et al. 21
In a previous study, teratospermic cats demonstrated an increased sperm output, mainly due to a reduced elimination of the defective spermatogenic cells via apoptosis. 22 In contrast, in this report, although the testicular volume of the teratospermic cat was slightly higher compared with the normospermic cats, the total sperm quantity was lower. Nonetheless, it is important to highlight that retrograde flow of sperm cells into the urinary bladder during natural mating or semen collection by an artificial vagina can occur in the domestic cat, 23 making it difficult, at times, to evaluate sperm production accurately.
Over the past few years possible causes for the occurrence of the Dag or Dag-like defect have been suggested and include a weakness in the outer dense fibers due to zinc excess, 24 premature release of hydrolytic enzymes from the distal droplet 19 or, most likely, a malformation in the mitochondrial sheath during late spermiogenesis.5,25 In this report, considering the fact that testicular sperm cells from the teratospermic cat already presented some structural changes in the midpiece, perhaps the origin of the Dag-like defect in the domestic cat might be related to a disturbance in mitochondrial sheath formation, as hypothesized previously in other species.3,5,18,25
Although abnormal epididymal secretions can lead to bent tail defect, in some cases the underlying cause might be a structural weakness caused by abnormal development in spermiogenesis. 5 The fact that the teratospermic cat from this report showed normal values for BZn, SPZn and serum testosterone, the latter being one of the main regulators of epididymal function, 26 also adds some support to our hypothesis that the Dag-like defect has a testicular origin. Furthermore, once a high percentage of the sperm cells affected by the Dag-like defect also showed macrocephalia, an overall disturbance in the process of spermatogenesis might be involved.
Interestingly, a slight decrease in the percentage of macrocephalic sperm cells with or without the Dag-like defect was found after epididymal transit, perhaps owing to the capacity of the cat epididymides to reduce morphologically abnormal spermatozoa. 10 Moreover, we observed an increase in coiling, folding and fracture of the midpiece after epididymal transit. 18 Epididymal sperm cells were retrieved in a medium that permitted the cells to start their motion and only then fixed in formol–saline for morphology assessment. For this reason, the finding of a similar percentage of the Dag-like defect between epididymal and ejaculated spermatozoa might be a consequence of gaining motility. It’s well-known that a normally developed mitochondrial sheath is essential in providing structural support to the axial fibers in the midpiece region during flagellar motion,3,5,18 which is not the case for the cells affected by the Dag-like defect.
In this case report, while no difference was observed for the BSe, lower SPSe was found for the cat presenting the Dag-like defect, even though all animals had received the same commercially-produced dry food for >1 year before selenium measurement. Selenium-containing proteins represent a major polypeptide of the mitochondrial capsule, which is a rigid structural complex that, during maturation in the epididymides, becomes stabilized by disulfide bonds and organizes the mitochondrial sheath around the flagellum. 27 Thus, selenium deficiency in males can lead to a reduction or loss of motility and to an increase in spermatozoa abnormalities, localized mainly in the midpiece.28–31 Recently, phospholipid hydroperoxide glutathione peroxidase (PHGPx), a selenocysteine-containing selenoprotein, has been proposed to play a dual role in spermatozoa, both protecting it against lipid peroxidation and forming the mitochondrial capsule. 32 Taking into account that (i) the negative effect of selenium deficiency upon sperm quality has been proposed to appear only at an extremely low selenium intake in other species, 29 (ii) the lack of information concerning the optimal selenium status regarding male fertility and (iii) the presence of normal BSe in this report, a correlation between the Dag-like defect occurrence and a low selenium concentration needs further investigation.
Considering that the tom cat of this report constantly displayed a high incidence of sperm tail defects, including the Dag-like defect, and no health or environmental disorders were detected, perhaps a genetic factor might be involved. Unfortunately, because both breeding and bloodline records from this tom cat are unknown, we cannot establish a hereditary basis for the Dag-like defect in cats, as seen in bulls.1,7,19 However, it is worth mentioning that teratospermia in felids can be linked to a reduction in genetic variability, which contributes to pleiomorphic sperm production. 20
Conclusions
To the authors’ knowledge, this is the first report of the Dag-like defect in the domestic cat. Furthermore, evidence indicates a testicular origin for this sperm defect, probably during late spermiogenesis, although an epididymal disturbance may be also associated.
Footnotes
Acknowledgements
The authors thank FAPESP for the financial support, and Royal Canin for the food supply.
Funding
The study was funded by the São Paulo Research Foundation (FAPESP).
Conflict of interest
The authors do not have any potential conflicts of interest to declare.