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Abstract

Objectives

The current study aimed to evaluate the relationship between specific seminal plasma components – cholesterol (CHOL), triacylglycerols (TAG) and total protein (PROT) concentrations – and semen quality in cats. A further aim was to determine the relationship between specific seminal protein bands and semen quality.

Methods

Thirteen toms, 2–5 years of age, were included. Semen collection was performed by electroejaculation every 4 weeks. Fifty-eight ejaculates were assessed for motility, velocity, volume, sperm concentration, total sperm count, viability, acrosome integrity, plasma membrane integrity and sperm morphology. Samples were divided into two groups: good semen quality (GSQ) and poor semen quality (PSQ). After evaluation, seminal plasma was separated from the sperm by centrifugation and stored at −20°C. CHOL, TAG and PROT concentrations were then assessed and seminal plasma protein profile was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).

Results

Seminal plasma CHOL and TAG concentrations, motility, velocity, sperm concentration, total sperm count and sperm morphology were significantly higher in GSQ cats compared with PSQ cats (P <0.01). Moreover, seminal plasma SDS-PAGE analysis showed an identifiable extra band exclusively in the GSQ group.

Conclusions and relevance

Data obtained in this study showed that seminal plasma CHOL and TAG concentrations and specific protein bands could be used to improve semen evaluation in toms. In this sense, the 14 kDa protein band could be a valuable marker for semen quality in the cat and should be further investigated. However, more studies are necessary to determine its relationship with fertility.

Keywords

Sperm seminal plasma electrophoresis seminal plasma biochemic sperm analysis

Introduction

Seasonal variations in semen quality (SQ), as well as the high variability in seminal samples between and within cats, has been widely documented.^1–4 However, information related to seminal plasma composition is scarce for tom cats.⁵ Further, the relationship between the biochemical composition of seminal plasma and SQ in cats has not been described yet. Moreover, in some induced ovulator species, it has been reported that ovulation is elicited by a seminal plasma protein component (ovulation-inducing factor [OIF]), identified as a neurotrophin nerve growth factor beta (β-NGF) in camelid species.⁶ OIF has also been described in koalas and rabbits,^7,8 suggesting that it could also be a constituent of feline seminal plasma. Recently, the presence of an OIF in feline seminal plasma has been suggested.⁹

The importance of cholesterol during spermatogenesis, in addition to being a component of the seminal fluid, has been described. In this way, it is known that cholesterol is required for the mass production of germ cells during spermatogenesis.^10–13 Garolla et al reported a great difference in the cholesterol and its oxidized derivatives in sperm membrane between fertile and infertile human primate males, suggesting a strict biochemical link relating testis function, sperm membrane status and males’ fertility potential.¹⁴ Also, a recent study reported that the total amount of cholesterol in the seminal plasma of men was positively associated with SQ.¹⁵Similarly, an association of the fatty acid composition of seminal plasma, SQ and fertility/infertility has been recorded in stallions and bulls.^16,17 Seminal plasma also contains triglycerides, which are essential for sperm metabolism, since their oxidation supplies the sperm with its energy requirements.¹⁸ A high concentration of triglycerides in seminal plasma is associated with good quality semen in bulls.¹⁹ The relationship between the lipid composition of seminal plasma, seasonality and SQ has been reported in bulls.¹⁷Likewise, a positive correlation between sperm motility, velocity and specific protein bands has been reported in bulls and dogs.^20,21Given the above findings, determination of biochemical parameters has been proposed as a predictive tool for quality semen evaluation in male animals and men.^17,22,23 Even though seminal biochemical parameters have been studied in men and in some domestic animals, in the domestic cat, there are few reports on this topic. It would be interesting to include biochemical determination as an SQ predictor in routine semen evaluation when tom cats are part of breeding programs in colonies or shelters.

Therefore, the aims of this study were: (1) to assess seminal plasma protein (PROT), cholesterol (CHOL) and triacylglycerol (TAG) concentrations and their relationship with SQ in tom cats; and (2) to study the seminal plasma protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to determine the relationship between specific protein bands and SQ. We hypothesized that certain seminal plasma biochemical parameters could be used as a tool to predict SQ in cats.

Materials and method

Experimental design

Animals

Thirteen mixed-breed toms, 2–5 years of age, were included. Tom cats were maintained in a controlled environment (room dimensions, 3.5 × 4.6 m), housed alone and fed with commercial cat food (pH control; Vital Can) and water ad libitum. An artificial illumination schedule was established with alternated 2-month photoperiod cycles to maintain SQ with 1400 lumen LED lamps giving 150–300 lux at floor level.²⁴ A physical examination was performed in all animals weekly, and behavioral, food and water intake, and fecal changes were recorded daily.

Animal care, housing and experimentation complied with the International Guiding Principles for Biomedical Research Involving Animals.²⁵ This study was approved by the Graduate School and the Animal Care and Use Committees of Laboratory Animals, Facultad de Ciencias Veterinarias, Universidad Nacional de La Plata (CICUAL #57-2-16T).

Semen sample collection

Semen collection was performed by electroejaculation according to the Howard technique.²⁶ Toms were anesthetized with a combination of xylazine (0.5 mg/kg IM Kensol; Köning) and ketamine (20 mg/kg IM Ketamina 50; Holliday-Scott). Briefly, each tom received a total of 80 stimuli divided into three sets (30, 30 and 20) with 2–3 mins of rest between sets. The first set consisted of 10 stimuli at 2 V, 10 at 3 V and 10 at 4 V. The second set consisted of 10 stimuli at 3 V, 10 at 4 V and 10 at 5 V. The third set consisted of 10 stimuli at 4 V and 10 at 5 V.²⁶ Semen samples were collected into a 1.5 ml prewarmed Eppendorf tube and immediately assessed. Samples were collected every 4 weeks for a period of 6 months (from June to November). A total of 58 ejaculates were evaluated.

Semen sample evaluation

Ten microliters of fresh semen were used to estimate motility (MOT; % motile) and velocity (VEL; 0–5) subjectively under an optic microscope equipped with a warming plate at × 400 magnification in 5–7 microscopic fields. The volume (VOL; µl) of the ejaculate was measured with the variable pipette (10–100 µl). Sperm concentration (SC; × 10⁶/ml) was evaluated with a Neubauer counting chamber by diluting 5 µl of semen in 1 ml of formol saline solution. SC multiplied by VOL delivers the total number of spermatozoa in the ejaculate (total sperm count [TSC]; × 10⁶). Sperm viability (VIA; % alive) was evaluated in 5 µl of semen using eosin/nigrosin staining. Sperm plasma membrane integrity (PMI; % intact) was additionally determined by carboxyfluorescein diacetate/propidium iodide (CFDA-PI) in 10 µl of semen.²⁷ Acrosome integrity (AI; % intact) was determined by fluorescein isothiocyanate-labeled Pisum sativum agglutinin (FITC-PSA) in 5 µl semen.²⁸ Sperm morphology (SM; % normal) was assessed by counting 150 spermatozoa at random under an optic microscope (magnification × 1000) in 5 µl of semen using Tinción 15 (Biopur, Rosario).

MOT, VEL and SC were used to perform an evaluation index of SQ. Each variable was categorized by terciles and ejaculates. All three variables in the highest tercile were classified as being of good semen quality (GSQ), and those ejaculates with all three variables in the lowest tercile were classified as being of poor semen quality (PSQ). From the total of 58 ejaculated evaluated, 28 were selected for the study (GSQ: n = 11; PSQ: n = 17). Each cat produced samples that were included in the GQS and PQS groups. Data regarding the SQ of GSQ toms agree with previous reports.^24,29,30 In the PQS group, sperm PMI was not evaluated by CFDA-PI because of insufficient sperm VOL.

After semen evaluation, seminal plasma was separated from the sperm by centrifugation (700 g for 10 mins), a drop of the supernatant was evaluated by microscopy to confirm the absence of cells, and sperm-free seminal plasma was stored at ‒20°C until used.²¹

Seminal plasma biochemical analysis

PROT, CHOL and TAG measurements were performed in seminal plasma (n = 58) using a Metrolab plus 1600 Clinical Analyzer (MM Instrumental Científico), with the Proti 2 and Colestat (Wiener) and Triglycerides kits (BioSystems) following the manufacturer’s instructions. Detection limits were: 0.02–12 g/dl (PROT); 0.0063–5 g/l (CHOL); and 1.6–600 mg/dl (TAG).

SDS-PAGE

The average total PROT concentration was 37.08 ± 6.62 mg/dl, and 5 µl of sperm-free seminal plasma was loaded per lane (average of total proteins: 1.9 µg in the GSQ group and 1.5 µg in the PSQ group). Seminal plasma was reduced and denatured in Laemmli buffer (62.5 mM TRIS-HCl, pH 6.8 [Sigma-Aldrich], 10% (v/v) glycerol [Sigma], 2% (v/v) SDS [Sigma], 5% beta-mercaptoethanol [Sigma] and 0.2% bromophenol blue) at 95ºC for 5 mins and centrifuged for 1 min at 3000 g. The low-molecular-weight marker (LMW-SDS Kit; GE Healthcare Life Sciences) was loaded in lane 1. Proteins were separated by electrophoresis on 4–15% acrylamide/bisacrylamide gel (SDS-PAGE) based on the protocol of Laemmli³¹ and transferred to polyvinylidene fluoride membranes using the Enduro Electrophoresis System (Labnet International). Protein bands were seen by exposing membranes with India ink stain (5 ml acetic acid 60.05 g/mol, 0.5 ml Pelikan Fount India drawing ink for fount pen [Pelikan 17 black], 500 ml in phosphate buffered saline ×1, 0.05% Tween20) for 5 h.³²

Statistical analysis

Data were analyzed by ANOVA using the ProcGlimmix of SAS (9.4) and Pearson’s correlations between all variables were estimated (ProcCorr, SAS 9.4). The variation of CHOL, TAG and PROT were explained by univariable regression models with SQ as the fixed predictor, and including cat as a random effect to take into account the repeated measures of ejaculates within cats. Least square mean differences between GSQ and PSQ were assessed in all models by using a Student’s t-test (ProcGlimmix SAS 9.4).³³

Results

Seminal parameters for the GSQ and PSQ groups are shown in Table 1. Seminal plasma CHOL and TAG concentrations were significantly higher in the GSQ group compared with the PSQ group (P <0.0001). However, there were no significant differences in seminal plasma PROT concentration (P >0.14). Semen MOT, VEL, SC, TSC and SM were significantly higher in the GSQ group compared with the PSQ group (P ⩽0.01). In contrast, there were no significant differences in semen VOL, VIA or AI (P >0.25).

Table 1

Seminal parameters of good semen quality (GSQ) and poor semen quality (PSQ) groups

Semen parameter	GSQ (n = 11)	PSQ (n = 17)	P value
Seminal plasma cholesterol (mg/dl)	18.97 ± 1.50	6.94 ± 1.21	<0.0001
Seminal plasma triacylglycerides (mg/dl)	101.77 ± 9.26	21.19 ± 7.45	<0.0001
Seminal plasma protein (mg/dl)	50.00 ± 7.35	33.33 ± 5.19	0.14
Motility (%)	89.82 ± 3.17	32.39 ± 2.65	<0.0001
Velocity (0–5)	4.96 ± 0.07	2.95 ± 0.06	<0.0001
Volume (µl)	106.57 ± 13.79	95.71 ± 11.32	0.48
Concentration (× 10⁶/ml)	137.27 ± 14.79	13.10 ± 11.89	<0.0001
Total sperm count (× 10⁶)	14.28 ± 1.32	1.28 ± 1.06	<0.0001
Viability (%)	70.27 ± 4.33	61.50 ± 5.86	0.25
Acrosome integrity (%)	56.50 ± 10.36	65.00 ± 17.95	0.72
Plasma membrane integrity (%)	70.02 ± 6.38	–	0.59
Sperm morphology (%)	69.95 ± 5.07	49.75 ± 6.56	0.01

Least squares means ± SE

The following significant and positive correlations were found: CHOL with TAG; CHOL and TAG with MOT, VEL, SC, TSC and SM (P <0.01; Table 2); and CHOL with PMI in the GSQ group (P <0.01; Table 2). In addition, the following significant and positive correlations were also found: MOT with VEL, SC, TSC and SM; VEL with SC, TSC and SM; SC with TSC; SC and TSC with VIA; and VIA with PMI (P <0.01; Table 2). In addition, the relationship between TSC and the amount of seminal plasma CHOL and TAG is shown in Table 3. However, no correlation between PROT with any seminal parameters was detected (P >0.10).

Table 2

Pearson’s correlations between seminal plasma biochemical evaluation and seminal parameters

	TAG	PROT	MOT	VEL	SC	TSC	VIA	AI	PMI	SM	VOL
CHOL	0.75†	0.01	0.53†	0.59†	0.53†	0.57†	0.20	−0.001	0.66†	0.45†	0.07
TAG		−0.16	0.59†	0.64†	0.53†	0.64†	0.23	0.03	0.20	0.44†	0.24
PROT			0.13	0.31	0.10	0.25	0.06	−0.35	−0.18	−0.14	0.18
MOT				0.78†	0.41†	0.44†	0.22	−0.12	0.32	0.70†	0.07
VEL					0.43†	0.49†	0.07	−0.14	0.31	0.49†	0.10
SC						0.90†	0.43†	0.30	0.42	0.26	−0.05
TSC							0.38*	0.33	0.46	0.19	0.26*
VIA								0.45*	0.70†	0.21	−0.09
AI									0.06	0.13	0.10
PMI										0.34	−0.10
SM											−0.29
VOL

P <0.05†P <0.01

CHOL = cholesterol (mg/dl); TAG = triacylglycerides (mg/dl); PROT = seminal plasma protein (mg/dl); MOT = motility (% motile); VEL = velocity (0–5); SC = sperm concentration (×10⁶/ml); TSC = total sperm count (×10⁶); VIA = viability (% alive); AI = acrosome integrity (% intact); PMI = plasma membrane integrity (% intact); SM = sperm morphology (% normal); VOL = volume (µl)

Table 3

Relationship between total sperm count and the amount of seminal plasma cholesterol (CHOL) and triacylglycerides (TAG) in good semen quality (GSQ) and poor semen quality (PSQ) groups

	GSQ (n = 11)	PSQ (n = 17)
Seminal plasma CHOL (µg)/1 ×10⁶ sperm	1.33 ± 0.11	52.74 ± 0.91
Seminal plasma TAG (µg)/1 ×10⁶ sperm	7.12 ± 0.65	16.10 ± 5.67

Seminal plasma protein electrophoresis clearly showed five protein bands in all samples corresponding to molecular weights of 20.1, 30.0, 45.0, 66.0 and 97.0 kDa. Only the seminal plasma of the GSQ group presented an extra band of 14.4 kDa (Figure 1).

Figure 1

Cat seminal plasma protein separation on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Seminal plasma samples were reduced, denatured and separated on 15% polyacrylamide gel and transferred to polyvinyldifluoride membranes stained with India ink. Lanes 1, 2 and 6 were loaded with 5 μl of seminal plasma of poor semen quality (1.8, 1.6 and 2 µg of total proteins, respectively). Lanes 3–5 and 7 were loaded with 5 μl of seminal plasma of good semen quality (1.8, 1.7, 2 and 2.1 µg of total proteins, respectively). Lane 8 was loaded with a low-molecular-weight marker (14.4–97.0 kDa). Note that the 14.4 band is present only in the good semen quality group

Discussion

In the current study, a relationship between seminal plasma TAG and CHOL concentrations and SQ was demonstrated. This is in agreement with previous studies in stallions and bulls.^16,17One of the biochemical changes associated with the capacitation process is an efflux of cholesterol from the plasma membrane leading to an increase in membrane fluidity and permeability to bicarbonate and calcium ions.³⁴ It has been shown previously that the in vitro incubation of bovine sperm with female reproductive tract fluids causes cholesterol loss.³⁵ Lost sperm plasma membrane cholesterol is then bound to lipoproteins or albumin in uterine and follicular fluid.^36,37 This cholesterol efflux from the sperm plasma membrane is crucial for sperm capacitation. In this regard, seminal plasma plays an important role in preventing sperm capacitation by supplying additional cholesterol to the plasma membrane.^38–40 As a result, cholesterol has been added to extenders to prevent freeze–thaw sperm damage, which may impair sperm MOT and fertilizing potential.^41,42 In bulls, a high concentration of seminal plasma CHOL has been found during the winter season, associated with GSQ, and in the summer season, PSQ was associated with reductions in seminal plasma CHOL and semen fatty acid concentrations.^17,19 Our findings are in agreement with those in bulls, since higher CHOL seminal plasma concentrations were associated with GSQ. Also, our results showed a significant and positive correlation between seminal plasma CHOL and spermatozoa PMI in the GSQ group, supporting the hypothesis that the high CHOL concentration in seminal plasma prevents sperm capacitation.In the current study, a positive and significant correlation was observed between seminal plasma CHOL and MOT; this finding is in agreement with previous reports in bulls.^17,19 A recent report in humans showed a positive association between total CHOL in seminal plasma and sperm MOT, SC, TSC and SM.¹⁵ Those findings in humans are in agreement with our findings in cats.

TAG are essential for sperm metabolism, as their oxidation supplies the sperm with its energy requirements, and play a role in the fertilizing ability of sperm.^18,19,43,44 Kacel and Iguer-Ouada observed an improvement in seminal quality and a higher triglyceride concentration in seminal plasma in roosters fed with a diet supplemented with olive oil vs roosters fed a standard diet.⁴⁵ In agreement with this previous study, we found better SQ in toms with a higher seminal plasma TAG concentration. Furthermore, a significant and positive correlation was observed between seminal plasma TAG and MOT, VEL, SC, TSC and SM. In this sense, the measurement of CHOL and TAG concentrations could be included in the conventional semen assessment in cats to estimate the ejaculate quality in tom cats.

Interestingly, the data in Table 3 show that the amounts of seminal plasma CHOL and TAG per million spermatozoa were higher in the PSQ group, which had a lower semen SC than the GSQ group. Even though seminal plasma CHOL and TAG concentrations in the PSQ group were lower than in the GSQ group, the low number of sperm/ml in the PSQ group led to a higher amount of seminal plasma CHOL and TAG per million sperm cells.

No correlation between seminal plasma PROT concentration and SQ was observed in dogs.²¹ In the current study, the mean seminal plasma PROT concentrations were similar to those reported previously in the cat.⁵ Zambelli et al also evaluated a protein profile in seminal cat plasma and found >30 protein bands with molecular weights that ranged from 3.5 to 200 kDa in electroejaculated and urethral catheterization samples. However, only eight protein bands were found in all samples, and three proteins (P200, P80, P28) were more concentrated in electroejaculated than in urethral catheterization samples.⁵ We clearly identified five protein bands that ranged from 20 to 97 kDa in all seminal plasma samples. In contrast to Zambelli et al, we found the 14.4 kDa protein band in ejaculates obtained by electroejaculation. Furthermore, this protein band was found exclusively in ejaculates corresponding to the GSQ group.

The study performed by Zambelli et al was carried out from January to June,⁵ while the tom cats in our study were placed in a controlled environment with an alternated photoperiod to maintain SQ.²⁴ Considering that sperm production and semen quality in tom cats is influenced by the season, differences between the studies could be related to different seasonal protein expression in cat seminal plasma.^1,2 In this regard, seasonal variation in SQ has been widely described in rams, and differences in seminal plasma protein composition were found between the breeding and non-breeding seasons.⁴⁶ Additionally, the analysis of seminal plasma by SDS-PAGE revealed that several protein bands were found only during the breeding season.^47,48 This seasonal variation in SQ and seminal plasma PROT have also been described in buffalo bulls. SQ (VOL, SC, VIA, AI, hypo-osmotic swelling test) was improved during the summer season. Also, the expression of seminal plasma PROT was increased in summer vs the winter and rainy seasons. In this sense, two high-molecular-weight protein bands were observed with SDS-PAGE only in the summer season.⁴⁹

During capacitation, cholesterol moves from the sperm membrane to soluble proteins acceptors; albumin has been described to be one of these acceptors.^10,50,51 In this regard, we observed an albumin-like protein band (66 kDa) in all seminal plasma samples. It could be that seminal plasma albumin in cats plays the same role as in humans.

Regarding seminal plasma proteins, SQ and fertility have been correlated with the presence of certain proteins bands in bulls, stallions and dogs.^20,21,52 In dogs, two protein bands (67 and 58.6 kDa) were positively correlated with seminal quality.²¹ In the same way, the 14.4 kDa protein band of cat seminal plasma was associated with GSQ. Therefore, the 14.4 kDa protein band could be used to improve semen evaluation in toms. Also, in llama seminal plasma a 14 kDa protein band was described as an OIF and later identified as β-NGF.⁶ Therefore, the 14.4 kDa protein band found in the cat seminal plasma of the GSQ group could be related to the presence of the OIF in domestic cats. This fact could also be related to semen fertility and the ability to induce ovulation in the queen. Further studies are necessary to understanding cats’ reproductive physiology.

In bulls, two protein bands (26 kDa and 55 kDa) were associated with high fertility, and the 16 kDa protein band was associated with low fertility.²⁰ In stallions, the 72 kDa protein band was positively correlated with fertility.⁵² In the current study, the fertility of the cats was unknown, so further studies are necessary to demonstrate the potential role of the 14.4 kDa protein band to predict fertility in toms.

In the current study, we used toms belonging to the cat colony from the School of Veterinary Medicine. However, it would be interesting to study seminal plasma PROT, TAG and CHOL over a wider age range to know the effect of age on seminal plasma composition.

Conclusions

To our knowledge, this is the first report to positively correlate certain seminal plasma components with semen quality in cats. Data obtained in this work showed that seminal plasma CHOL and TAG should be used to improve semen evaluation in cats. However, more studies are necessary to determine their relationship with fertility. Furthermore, the 14.4 kDa protein band found in the seminal plasma of the GSQ group could be a valuable SQ marker and should be investigated further. Also, future studies need to be performed to determine if the 14.4 kDa protein band is related to the presence of β-NGF in cat seminal plasma, as it has been described in other induced ovulator species. Finally, the results of this topic in domestic cats could also be valuable for use in wild felids.

Footnotes

Acknowledgements

We thank Drs MC Venturini and G Moré, Laboratorio de Inmunoparasitología, Faculdad de Ciencias Veterinarias Universidad Nacional de La Plata for the use of their electrophoresis equipment, and Vital Can for providing the commercial premium cat food.

Accepted: 23 October 2019

Conflict of interest

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

This study was supported in part by Universidad Nacional de La Plata (UNLP) grant V11/230 to RLS and MAS and by UNLP PPID grant V009 to RNF.

Ethical approval

This work involved the use of experimental animals; or involved the use of non-experimental animals (owned or unowned) outside of established internationally recognized high standards (‘best practice’) of individual veterinary clinical patient care. The study therefore had ethical approval from an established committee as stated in the manuscript.

Informed consent

Informed consent (verbal) was obtained from the legal custodian of all animal(s) described in this work for the procedure(s) undertaken. For any animals or humans individually identifiable within this publication, informed consent for their use in the publication (verbal or written) was obtained from the people involved.

ORCID iD

María A Stornelli

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Relationship between semen quality and seminal plasma cholesterol,triacylglycerols and proteins in the domestic cat

Abstract

Objectives

Methods

Results

Conclusions and relevance

Keywords

Introduction

Materials and method

Experimental design

Animals

Semen sample collection

Semen sample evaluation

Seminal plasma biochemical analysis

SDS-PAGE

Statistical analysis

Results

Discussion

Conclusions

Footnotes

Acknowledgements

Conflict of interest

Funding

Ethical approval

Informed consent

ORCID iD

References