Therapeutic effect of single intra-testicular injection of lyophilized xenogenous and allogeneic mesenchymal stem cell-derived extracellular vesicles on oligozoospermia in male dogs.

doi:10.21203/rs.3.rs-4861862/v1

Download PDF

Research Article

Therapeutic effect of single intra-testicular injection of lyophilized xenogenous and allogeneic mesenchymal stem cell-derived extracellular vesicles on oligozoospermia in male dogs.

https://doi.org/10.21203/rs.3.rs-4861862/v1

This work is licensed under a CC BY 4.0 License

You are reading this latest preprint version

Oligozoospermia is the most common cause of infertility in male canine reproduction. Practitioners look for specific therapies to solve this problem. So, the aim of the present investigation is to study for the first time the impact of a single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on the oligospermia of male dogs. Dogs were randomly allocated into three groups. Group I is the control group (n = 5), without any treatment; Group II is the lyophilized canine MSCs-derived EVs (n = 5); and Group III is the lyophilized human MSCs-derived EVs (n = 5). Dogs were subjected to semen collection, blood collection, and Doppler examination. The assessment was performed on day 0 before injection, and the examination was extended to 8 weeks. The results showed that there was a notable (P ˂ 0.01) increase in testicular volume in either the canine or human MSCs-derived EVs group from the 6^th to the 8^th weeks of the study as compared to Day 0. The maximum volume of right or left testis in dogs treated with either canine or human MSCs-derived EVs was observed at 8 weeks post injections compared to Day 0 before treatments. The Doppler indices (PSV and EDV) were significantly improved in dogs treated with canine or human MSCs-derived EVs. The maximum values of PSV and EDV were attained in the 8^th week following injection. However, the PI and RI of the testicular arteries were significantly decreased as compared to the Day 0 and control group. The human MSCs-derived EVs group had lower values of RI and PI than the canine MSCs-derived EVs group. The single intra-testicular injection of lyophilized canine MSCs-derived EVs resulted in a highly significant (P<0.001) increase in the sperm cell concentration, motility, live/dead ratio, and normal spermatozoa compared to those obtained in lyophilized human MSCs-derived EVs. In addition, the canine MSCs-derived EVs group showed higher values (P<0.001) of testosterone levels from 2^nd to 8^th weeks than those in the human MSCs-derived EVs group. The reverse trend was observed for NO levels. In conclusion, a single intra-testicular injection of MSCs-derived EVs could improve testicular vascularization by increasing Doppler velocities. In addition, it could improve semen quality, steroids (testosterone), and nitric oxide.

Canine

Human

Lyophilized

Mesenchymal stem cells

Extracellular vesicles

Semen

Doppler.

Dogs may be bred as long as they are mostly fertile, and it is still important to preserve this species' capacity for reproduction [1]. Before a male's reproductive life begins, the quality of its semen can be used to forecast the male's probable fertility [2]. Breeders and practitioners currently focus a great deal of attention on the reproductive failure of the canine species, and they search for targeted therapies to address this issue. Several cases of presumed sub- and infertility are caused by male factors, particularly when the patient presents with an altered semen analysis [3, 4]. The infertility of male dogs is still a subject of little understanding. Poor semen quality is one of the many issues that might cause it [5–8]. Reduced spermatozoa production by the testes during the course of reproductive life is a notable clinical disorder known as oligozoospermia. Although its pathogenesis is yet unknown, oligozoospermia is thought to be one of the most common clinical reasons for male infertility in humans [9, 10].To date, no single medication has been found to be effective in treating the condition. Much study on using natural sources to treat oligozoospermia has been conducted in the past 10 years [11]. According to recent research, 15–20% of male dogs raised for breeding have subfertility issues [12]. Seminal analysis is used to diagnose the main issues, which are connected to low-quality semen, much like in humans. According to Rijsselaere et al. [13] and Domosławska et al.[5], the majority of assessed motility parameters, sperm concentration, and the percentage of spermatozoa with normal morphology are all considerably lower in infertile dogs than in fertile dogs. Sperm cell concentration is typically assessed for evaluating semen in dogs, although it is not a very useful indication because it is inversely related to ejaculate volume [2]. In recent years, many researchers have reported the impact of extracellular vesicles (EVs) on animal reproduction and fertility [14, 15]. Extracellular vesicles (EVs) are membrane-bound nanovesicles released by cells that are capable of carrying cargo between cells as a means of intercellular communication. This cargo includes proteins, DNA, and RNA. Based on their biogenesis or release mechanisms, many EV forms, including microvesicles (MVs), exosomes, oncosomes, and apoptotic bodies, have been described. Microvesicles originate from the plasma membrane directly, possessing cytoplasmic cargo and ranging in size from 100 nm to 1 µm [16]. According to El Andaloussi et al. [17] and Cocucci and Meldolesi [18], multivesicular bodies produce smaller vesicles, known as exosomes, with diameters ranging from 40 to 120 nm, when they fuse with the plasma membrane. Exosomes act as both endocrine and paracrine mediators in interactions with neighboring cells, which is crucial for cell-to-cell communication [19]. However, dying cells produce vesicular apoptotic bodies (50 nm–2 µm), which can vary in composition between biofluids and, under some circumstances, be more common than exosomes or MVs [17, 20]. In addition, membrane protrusions can give rise to large EVs called oncosomes (1–10 µm), which, unlike their non-transformed counterparts, are preferentially generated by malignant cells [21]. EVs can be found in a variety of bodily fluids, including breast milk, urine, saliva, and blood [22]. The EVs circulate freely in all bodily fluids until they attach to target cells that are close to or far from their site of origin. Once the target cells get their cargo, they may change the way they operate [23]. Thus, EVs are crucial for cell-to-cell communication and have a function in a variety of pathologies, such as immunological responses, cancer, neurological diseases, and physiological reproductive processes such as placenta development and embryo implantation [24–26]. Although seminal plasma was among the first organic fluid EVs to be isolated and described, it continues to be one of the least studied [27]. Compared to blood or cerebrospinal fluid, the concentration of EVs in seminal plasma is comparatively greater [28]. Male accessory glands, specifically the vesicular and prostate glands, as well as functional cells of the testis, epididymis, and vas deferens, release seminal EVs (sEVs) [29]. Seminal EVs participate in a variety of reproductive processes by binding to and exchanging active molecules with mature sperm and endometrial epithelial cells [26, 29]. Therefore, sperm motility, capacitation, and acrosome reaction, all functional sperm characteristics necessary for fertilization, would be regulated by sEVs. Additionally, normozoospermic and non-normozoospermic males have different cargo loads on sEVs [30]. By regulating the uterine immune response, seminal EVs would also aid in the safe passage of spermatozoa via the female genital canal [31].Extracellular vesicles (EVs) originating from stem cells have the capacity to trigger angiogenesis in endothelial cells [32], suppress cell death and promote cell proliferation [33], transmit immunomodulatory signals [34], and reprogram cells necessary for tissue regeneration [35]. The drawbacks of fresh exosomal solutions include the need to isolate and re-inject the solution into the same individual, their short shelf life, and the lengthy preparation period. Because of this, using preserved EVs was required in order to make them suitable for clinical application. Freeze drying, also known as lyophilization, is a technique used to preserve perishable items by dehydrating samples to delay degradation. Water is necessary for the development of microbes and the action of enzymes. Lyophilization is the best method for preserving thermosensitive substances like EVs, vaccines, viruses, proteins, peptides, and colloidal carriers because it is a two-step preservation process that involves freezing water and then removing it under pressure (4.579 mm of Hg) and at a temperature below 0.0099°C [19]. The research states that low sperm concentration and/or function are the main reasons impeding breeding programs. Consequently, several studies investigated different protocols to enhance semen quality in veterinary and human medicine using a specific daily intake of micronutrients as supplements [36–39]. For dogs, not much information is available [40, 41]. Furthermore, to the best of the authors' knowledge, there is no information available for dogs about the impact on semen quality of a single intra-testicular injection of lyophilized xenogenous mesenchymal stem cell-derived extracellular vesicles (human MSCs-derived EVs) or allogeneic mesenchymal stem cell-derived extracellular vesicles (canine MSCs-derived EVs). Therefore, in oligozoospermic dogs, the current study examined this influence on semen quality and testicular blood flow.

Experimental Location and Ethical Approval

This study was conducted at the dog stud of the Department of Theriogenology, Faculty of Veterinary Medicine, Cairo University (latitude 30°01' N; longitude 31°21' E), from October 1, 2023, to December 30, 2023. The study received ethical approval from the institutional animal care committee of the Faculty of Veterinary Medicine, Cairo University (Approval No.: Vet CU 18042024892)

2.1. Animals and management

Fifteen stray male dogs (30 ± 0.5 kg BW, age: 2 ± 0.5 years) were included in this study as, according to semen collection and evaluation, all of the male dogs had oligozoospermia. Dogs were randomly allocated into three groups. Group I is the control group (n = 5), without any treatment; Group II is the lyophilized canine MSCs-derived EVs (n = 5); and Group III is the lyophilized human MSCs-derived EVs (n = 5). Every animal involved in the study was kept inside, given regular exercise, a commercial diet consisting of cereals such as rice bran, lipids, vegetables, and vitamins, and allowed unlimited access to water. After the clinical examination, the male genital organs were ultrasonographically scanned for all of the males in the study. [42, 43].

2.2. Experimental design

In order to compare the effects of a single intra-testicular injection of lyophilized canine and human MSC-derived EVs on testicular hemodynamics and semen pictures to other non-injectable animals serving as controls, males were examined routinely (every two weeks, twice a month) at weeks 0, 2, 4, 6, and 8. Dogs were subjected to semen collection, blood collection, and Doppler examination. The assessment was performed on day 0 before injection, and the examination was extended to 8 weeks.

2.3. Source of lyophilized canine and human MSCs-derived EVs

Lyophilized canine and human mesenchymal stem cells (MSCs)-derived EVs powder was purchased from Bioluga® Canada, which was reconstituted in 4 ml of distilled water and mixed till obtaining a homogenous gel. Each 1 ml of the gel contained exosomes derived from 0.5×106 MSCs.

2.4. Single intra-testicular injection of lyophilized canine and human mesenchymal stem cell-derived extracellular vesicles

Using a sterile 21-gauge needle, each intra-testicular injection was carried out. The needle was withdrawn from the proximal end to the distal end, depositing the gel along the entire route by linear infiltration. The needle was directed from the caudoventral aspect of each testis, about 1 cm from the epididymal tail, towards the dorsocranial aspect of that testis. Two milliliters of the EVs solution were injected once into each testicle.

2.5. Ultrasonographic evaluation

Using the EXAGO Doppler ultrasound device (EXAGO, Echo Control Medical, Angoulême, France) with a linear array transducer with 5- to 9-MHz capability, ultrasound examinations were performed on the right and left testis of each dog. Dorsal recumbency was used to position the dogs, and the transducer was initially placed on the lateral surface of the testis after acoustic gel was applied to the skin. Testicular length and width were measured using longitudinal and transverse B-mode imaging, with the mediastinum used as a reference point. The formula for an ellipsoid, V = length x width x height x 0.5236, was used to calculate the testicular[44].

2.6. Measurement of the testicular artery Doppler parameters

All Doppler velocimetry measurements, such as peak systolic velocity (PSV; cm/sec), end-diastolic velocity (EDV; cm/sec), and both Doppler indices expressed by the resistance index (RI) and pulsatility index (PI) that are most frequently used, were determined from the three best continuous waves with complete systolic and diastolic endpoints [45, 46]. The distal branch of the testicular artery was identified using image-specific waves[47].

2.7. Semen collection and evaluation

It was best to do this procedure with an estrous bitch nearby. At the level of the bulbus glandis, the dog's penis was first vigorously rubbed through the prepuce until a partial erection appeared. The penis was then squeezed between the thumb and index finger, and pelvic thrusting was used to obtain a full erection. Following the collection of the first two fractions, the semen samples were sent to the lab for further analysis. Falcon tubes were used to collect all of the semen samples (50 ml; two collections were made after 28, 60 days following therapy, and one collection occurred prior to the intra-testicular injection of EVS solution). The concentration of sperm cells was determined using a hemocytometer and a light microscope[8]. Using a phase-contrast microscope with a heated stage (37°C) and 200× magnification, the percentage of motility of the collected sperm was subjectively evaluated to the closest 5% immediately after collection [48]. A sample was checked by two observers each, and there was never more than 5% fluctuation within a sample. Using dried smears stained with eosin/nigrosin, the percentage of live and dead spermatozoa was estimated [49]. About 100–200 spermatozoa under 100× (oil immersion) were counted on a stained semen slide to evaluate abnormalities. Only count free heads, not free tails[6].

2.8. Blood sampling and hormonal assaying

An hour following intravenous administration of Receptal® (GnRH analogue) at a dose of 0.4 µg/kg B.W., blood samples (2 ml) were drawn from the cephalic vein [50]. Centrifugation at 3,000 g for 5 minutes at room temperature obtained the serum [51]. Serum samples were stored at 20°C before being used for hormonal analysis. Using commercial enzyme immunoassay kits (ELISA), the blood testosterone level was determined, as described by Taya [52]. Nitric oxide (NO) was measured in serum samples using a spectrophotometer set at 540 nm, NO was measured using commercial kits (nitric oxide kit, Bio-diagnostic, Dokki, Egypt).

2.9. Statistical Analyses

All data are presented as the mean ± standard error of the mean and were first checked for normality by the Kolmogorov-Smirnov test. Repeated measures ANOVA test was used for discrimination of the differences among the means in the studied time points, and finally, the Bonferroni post hoc test. GraphPad Prism5 software was used for all the statistical assessments studied. P < 0.05 was considered statistically significant.

3.1. B-mode ultrasonic findings of volume of dog testes

Table 1 describes the influence of a single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on the testicular volume. At the beginning of the experiment, no significant change in testicular volume could be detected among the different groups. There was a notable (P ˂ 0.01) increase in testicular volume in the canine and human MSCs-derived EVs groups from the 6th to the 8th weeks of the study as compared to Day 0. The maximum volume of right or left in dogs treated with either canine or human MSCs-derived EVs was observed at 8 weeks post injections compared to Day 0 before treatments. Furthermore, a single intra-testicular injection of either canine or human MSCs-derived EVs resulted in a significant increase (P ˂ 0.01) in testicular volume when compared to the control group at the 6th week. In addition, no significant difference existed between groups treated with either canine or human MSCs-derived EVs (Fig. 1).

Table 1

Effect of single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on the volume of testes (cm³) (Mean ± SEM).
		Duration of study
Groups	Traits	0 week	2nd weeks	4th weeks	6th weeks		8th weeks
	Volume of Right testes	8.81 ± 0.44^a	9.02 ± 0.45^a	8.97 ± 0.48^a	9.28 ± 0.45^a		9.3 ± 0.45^a
Control
	Volume of Left testes	8.91 ± 0.19^a	9.02 ± 0.21^a	8.93 ± 0.18^a		9.29 ± 0.30^a		9.13 ± 0.27^a
	Volume of right testes	9.06 ± 0.48^ac	10.25 ± 0.14^ac	10.77 ± 0.19^ac		11.77 ± 0.39^ac		11.95 ± 0.21^bd
Canine EVs	Volume of left testes
	Volume of left testes	8.67 ± 0.13^ac	8.72 ± 0.24^ac	9.80 ± 0.25^ac		11.90 ± 0.20^ac		12.11 ± 0.16^bd
	Volume of right testes	8.41 ± 0.09^ac	10.47 ± 0.72^ad	11.09 ± 0.91^ad		12.06 ± 0.97^bd		12.78 ± 0.95^be
Human EVs	Volume of left testes
	Volume of left testes	8.91 ± 0.19^ac	10.41 ± 0.55^ad	10.01 ± 1.04^ad		12.25 ± 1.18^be		12.58 ± 1.07^be
Mean with different superscripts (a, b) within the same column, (c, d, e) within the same row were significantly different at P < 0.01.

3.2. Testicular hemodynamics

The effect of a single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on the testicular blood flow is shown in Table 2. The obtained results showed that the blood flow of the testes treated with either canine or human MSCs-derived EVs exhibited significantly higher (P < 0.001) EDV values at 2 weeks until the end of the study (8 weeks) than those seen on day 0 before treatments. The maximum value was attained in the 8th week following injection. On the other hand, the treated dogs showed a substantial increase in PSV from the 2nd and 4th weeks following human and canine MSCs-derived EVs injection, respectively, when compared to the control groups. Both PSV and EDV increased significantly (P < 0.001) at the 8th week following injections in both treated groups. However, no significant difference existed between the treated groups in PSV and EDV values. The dogs treated with human or canine MSCs-derived EVs had lower values of the RI and PI (P < 0.001) at the 2nd to 4th week following injection, respectively, than those obtained in the control groups (Fig. 2). Furthermore, the dogs in the human MSCs-derived EVs group had lower values of the RI and PI (P < 0.001) from the 2nd week until the 8th week than those in the canine MSCs-derived EVs group (Fig. 3).

Table 2

Effect of single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on testicular hemodynamic (Mean ± SEM).
		Duration of study
Groups	Measures	0 week	2nd weeks	4th weeks	6th weeks	8th weeks
	PSV	10.50 ± 0.43^a	10.80 ± 0.37 ^a	11.04 ± 0.48 ^a	11.58 ± 0.16 ^a	11.45 ± 0.15 ^a
	EDV	2.30 ± 0.08 ^a	2.55 ± 0.09 ^a	2.47 ± 0.14 ^a	2.38 ± 0.13 ^a	2.19 ± 0.07 ^a
Control	RI	0.77 ± 0.00 ^a	0.76 ± 0.01 ^a	0.77 ± 0.00 ^a	0.77 ± 0.00 ^a	0.76 ± 0.00 ^a
	PI	1.64 ± 0.04 ^a	1.58 ± 0.08 ^a	1.61 ± 0.05 ^a	1.60 ± 0.06 ^a	1.64 ± 0.04 ^a
	PSV	10.24 ± 0.34 ^ad	11.60 ± 0.51 ^ad	14.82 ± 0.97^be	16.76 ± 0.43 ^be	20.00 ± 0.40 ^be
	EDV	2.54 ± 0.21 ^ad	4.14 ± 0.30 ^be	4.47 ± 0.37 ^be	7.12 ± 0.21 ^be	7.69 ± 0.25 ^be
Canine EVs	RI	0.77 ± 0.01 ^{a d}	0.76 ± 0.01 ^{a d}	0.69 ± 0.01 ^{b e}	0.67 ± 0.01 ^{b e}	0.62 ± 0.01 ^{b f}
	PI	1.64 ± 0.05 ^{a d}	1.61 ± 0.06 ^{a d}	1.33 ± 0.03 ^{b e}	1.32 ± 0.02 ^{b e}	1.16 ± 0.03 ^{b e}
	PSV	10.44 ± 0.33 ^ad	13.24 ± 0.75 ^be	16.02 ± 0.50^bf	17.30 ± 0.41^bf	18.26 ± 0.20 ^bf
Human EVs	EDV	2.82 ± 0.20 ^ad	4.60 ± 0.35 ^be	5.34 ± 0.38 ^be	7.38 ± 0.49 ^cf	8.60 ± 0.21 ^bg
	RI	0.78 ± 0.00 ^{a d}	0.69 ± 0.01 ^{b e}	0.58 ± 0.01 ^{c f}	0.54 ± 0.01 ^{c g}	0.47 ± 0.01 ^{b h}
	PI	1.64 ± 0.04 ^{a d}	1.27 ± 0.06 ^{b e}	1.00 ± 0.03 ^{c f}	0.89 ± 0.06 ^{c f}	0.69 ± 0.26 ^{b g}
Mean with different superscripts (a, b, c) within the same column, (d, e, f, g) within the same row were significantly different at P < 0.001.

3.3. Semen analysis

The data revealed that all semen parameters, including sperm cell concentration, motility, live, and normal spermatozoa, increased significantly (P < 0.001) from the day 28 value, which continued to attain the maximum value at day 60 after treatment in the canine and human MSCs-derived EVs group when compared to the control and Day 0 at the start of the experiment, as shown in Table 3. It was clear that the single intra-testicular injection of lyophilized canine MSCs-derived EVs resulted in a highly significant (P < 0.001) increase in the sperm cell concentration, motility, live, and normal spermatozoa compared to those obtained in lyophilized human MSCs-derived EVs.

Table 3 Effect of single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on sperm cell concentration (x10⁶/ml), motility%, live /dead ratio and abnormalities % in dogs (Mean ± SEM).

		Duration of study

Groups	Measures	Day 0	Day 28	Day 60


	Sperm conc. (x10⁶/ml)	35.4±0.50^a	35.0 ±0.31^a	34.8±0.37^a
	Sperm conc. (x10⁶/ml)
Control	Motility %	49±1.87^a	48 ±1.22^a	48±1.23^a
	L/D ratio	86.6±0.81^a	89.0±1.18^a	89.4±0.87^a
	Abnormalities %	11.6±0.50 ^a	11.4±0.40^a	11.6±0.50^a
	Sperm conc. (x10⁶/ml)	35.0±0.32^ad	194.4 ± 1.91^be	353.4±1.53^bf
	Sperm conc. (x10⁶/ml)
Canine EVs	Motility %	48.6±0.98^ad	62.8 ±1.16^be	82.8±1.16^bf
	L/D ratio	89.0±1.18^ad	91.4±0.75^ad	95.4±0.51^be
	Abnormalities %	15.4±0.51^ad	11.6±0.51^be	8.0±0.62^bf
	Sperm conc. (x10⁶/ml)	35.0±0.31^ad	126.0±0.65^ce	154.2±2.15^c^f
	Sperm conc. (x10⁶/ml)
Human EVs	Motility %	48.6±0.97^ad	62.2±1.01^be	72.6±1.12^cf
	L/D ratio	89.0±1.18^ad	93.6 ±1.02^b^e	94.0±0.70^bf
	Abnormalities %	11.6±0.50^ad	8.4±0.50^c^e	7.8±0.37^be

Mean with different superscripts (a, b, c) within the same column, (d, e, f) within the same row were significantly different at P<0.001.

3.4. Serum testosterone and NO levels.

All dogs, either in the lyophilized canine or human MSCs-derived EVs group, had significantly higher testosterone and NO levels (P < 0.001) at 2nd to 8th weeks following injection than those obtained at day 0 before treatment and in the control group (Table 4,5). In addition, the canine MSCs-derived EVs group showed higher values (P < 0.001) of testosterone levels from the 2nd to the 8th week than those in the human MSCs-derived EVs group (Table 4). The reverse trend was observed for NO levels (Table 5).

Table 4

Effect of single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on the serum level of Testosterone (ng/ml) in dogs (Mean ± SEM.).
	Duration of study
Groups	0 week	2nd weeks	4th weeks	6th weeks	8th weeks
Control	2.72 ± 0.05^a	2.28 ± 0.07 ^a	2.29 ± 0.07 ^a	2.29 ± 0.07 ^a	2.31 ± 0.06 ^a
Canine EVs	2.80 ± 0.05 ^ad	4.62 ± 0.10^be	4.70 ± 0.07 ^be	4.80 ± 0.07 ^be	5.00 ± 0.07 ^bf
Human EVs	2.65 ± 0.00 ^ad	2.69 ± 0.00 ^cd	2.70 ± 0.00 ^cd	3.46 ± 0.01 ^ce	3.66 ± 0.01 ^ce
Mean with different superscripts (a, b, c) within the same column, (d, e, f) within the same row were significantly different at p < 0.001.

Table 5

Effect of single intra-testicular injection of lyophilized canine and human MSCs-derived EVs on the serum level of nitric oxide (µmol/ml) in dogs (Mean ± SEM.).
	Duration of study
Groups	0 week	2nd weeks	4th weeks	6th weeks	8th weeks
Control	32.22 ± 0.19^a	32.64 ± 0.16 ^a	32.50 ± 0.15 ^a	33.10 ± 0.25 ^a	33.26 ± 0.25 ^a
Canine EVs	32.86 ± 0.26 ^ad	37.3 ± 0.31^be	39.2 ± 0.24 ^bf	44.0 ± 0.30 ^bg	45.5 ± 0.20 ^bh
Human EVs	32.8 ± 0.12 ^ad	33.9 ± 0.33 ^ce	41.3 ± 0.36 ^cf	63.1 ± 0.19 ^cg	68.6 ± 0.28 ^ch
Mean with different superscripts (a, b, c) within the same column, (d, e, f, g, h) within the same row were significantly different at p < 0.001.

Due to canine subfertility and infertility are common problems in small animal reproduction that have a significant financial impact on the dog breeding business [53]. Numerous stud dogs have different reproductive problems that are usually treatable if detected early [54]. Protocols to enhance sperm quality have been suggested by a number of studies [41, 55]. Prostatic and testicular problems are the main causes of infertility in male dogs [5]. All of the oligozoospermic dogs in our study, however, were clinically healthy, and their owners were unable to identify the causes of their dogs' low semen quality. Remarkably, according to Johnson et al. [49], 70% of cases of male infertility are still idiopathic. Therefore, the goal of our study was to investigate the effects of a single intra-testicular injection of lyophilized canine or human MSCs-derived EVs on testicular hemodynamics and semen quality in order to treat oligozoospermia. In comparison to controls and the beginning of the trial, our results showed a substantial (P < 0.001) increase in sperm concentration, motility, and live/dead ratio starting on day 28 and reaching peak levels at day 60. This quick recovery is consistent with dogs' 60-day spermatogenesis period [56]. Previous studies have highlighted the beneficial role of EVs in male fertility, including enhancing sperm function and facilitating successful fertilization [57–61]. Extracellular vesicles play a crucial role in controlling the functions of seminal fluid and sperm in both healthy and pathological reproduction. EVs are produced by a variety of male reproductive tract cells, such as the testis, prostate, and epididymis. They are abundant and include regulatory elements that are necessary for the biology of the female reproductive system after mating as well as sperm function. They are essential for the maturation and preservation of post-testicular sperm in all mammalian species since they enhance sperm capacitation, acrosome reaction, motility, and fertilization processes. To the best of our knowledge, this study is the first to investigate how testicular hemodynamics in oligozoospermic dogs are affected by a single intra-testicular injection of lyophilized canine or human MSCs-derived EVs. We found that injecting EVs increased blood velocity indices (PSV and EDV) and decreased PI and RI of the testicular arteries, consequently enhancing testicular vascular perfusion. These changes suggest improved oxygen and nutrition delivery to the testis and decreased blood flow resistance [62, 63]. Additionally, nitric oxide (NO) and testosterone levels in treated dogs were considerably greater (P < 0.001) than in controls and pre-treatment groups, according to our findings. Given that testosterone is a key regulator of the structural morphology and proper physiology of seminiferous tubules [64], the high concentration of testosterone produced a notable improvement in semen quality, which was seen in both groups receiving EVs treatment. In the current investigation, testosterone levels were greater in dogs treated with lyophilized canine MSCs-derived EVs from 2 to 8 weeks after therapy than in dogs treated with lyophilized human MSCs-derived EVs. This may be attributed to the beneficial effects of allogeneic type of EVs. While NO levels were significantly higher in dogs treated with lyophilized human MSCs-derived EVs than in other groups. Regarding vascular perfusion, NO hinders platelet aggregation and prevents platelet adhesion with endothelial cells, thus playing a direct role in the initiation of the vasodilation process in the reproductive system, as previously described in dogs [65] and rams [66].

To the best of our knowledge, this is the first study to use lyophilized canine or human MSCs-derived EVs in the treatment of oligozoospermia in dogs. This data is significant for improving male dog productivity and reproductive patterns. In conclusion, a single intra-testicular injection of lyophilized MSCs-derived EVs could improve testicular vascularization by increasing Doppler velocities. In addition, it could improve semen quality, steroids (testosterone), and nitric oxide.

Acknowledgements

Not applicable.

Authors’ contributions

Alaa Mohamed and Mohamed Fathi designed the protocol and performed the practical work (Semen evaluation, doppler and ultrasonographic scanning). Mohamed M. Bahr prepared and injected the lyophilized canine and human mesenchymal stem cell-derived extracellular vesicles. K. H. El-Shahat and Ashraf A. Shamaa were included in supervision, methodology and drafting the paper. All authors drafted the manuscript, reviewed it, and approved the last version of the manuscript.

Availability of data and materials

All data collected or analyzed during this study are included in this published paper.

Ethics approval and consent to participate

All procedures on animals were approved by the Institutional Animal Care and Use Committee at the Faculty of Veterinary Medicine Cairo University with a number: Vet CU18042024892.All methods were carried out in accordance with relevant guidelines and regulations. All methods are reported in accordance with ARRIVE guidelines Not applicable.

Consent for publication

Not applicable.

Competing interests

There are no conflicts of interest to declare.

Funding

This article was supported by the Department of Graduate Studies, faculty of veterinary medicine, Cairo University in Egypt.

Nagashima JB, Songsasen N. Canid Reproductive Biology: Norm and Unique Aspects in Strategies and Mechanisms. Animals. 2021;11(3):653. https://doi.org/10.3390/ani11030653
Kustritz MR. The value of canine semen evaluation for practitioners. Theriogenology. 2007; 68(3):329-337. http://dx.doi.org/10.1016/j.theriogenology.2007.04.017
De Celis R, Pedrón-Nuevo N, Feria-Velasco A. Toxicology of male reproduction in animals and humans. Arch Androl. 1996; 37(3):201-218. http://dx.doi.org/10.3109/01485019608988523
Agarwal A, Prabakaran SA. Mechanism, measurement, and prevention of oxidative stress in male reproductive physiology. Indian J Exp Biol. 2005; 43(11):963-974. https://nopr.niscpr.res.in/handle/123456789/23266
Domosławska A, Zduńczyk S, Niżański W, Janowski T. Assessment of semen quality in infertile dogs using computer-assisted sperm analysis by the Hamilton-Thorne Semen Analyser. Journal of Veterinary Research. 2013;57(3):429-432. http://dx.doi.org/10.2478/bvip-2013-0074
Feldman E, Nelson R. Canine and Feline Endocrinology and Rerpoduction. WB Saunders. Philadelphia, USA. 1996:71-73. https://www.cabidigitallibrary.org/doi/full/10.5555/19962215526
Fontbonne A. Infertility in male dogs: recent advances. Rev Bras Reprod Anim. 2011;35(2):266-273.
Mickelsen W, Memon M, Anderson P, Freeman D. The relationship of semen quality to pregnancy rate and litter size following artificial insemination in the bitch. Theriogenology. 1993; 39(2):553-560. http://dx.doi.org/10.1016/0093-691X(93)90397-N
Haslett, C., E. Chilvers, N. Boon , N.Colledge, 2002. Davidson’s Principles and Practice of Medicine, Churchill Livingstone, Edinburgh, p. 711. https://scholar.google.com/scholar?cluster=7001562042251415896&hl=en&as_sdt=2005&sciodt=0,5
10. Pramanik, D., 2007. Principles of Physiology.Kolkata: Academic Publishers, pp. 370–374. https://books.google.com/books?hl=en&lr=&id=XLNoc6vpVOYC&oi=fnd&pg=IA2&dq=Pramanik,+D.,+2007.+Principles+of+Physiology.&ots=e7PArq4rDt&sig=ow_S0CX4OFnkGhVc-gD3nOin3v4
Mukram MA, Rafiq M, Anturlikar SD, Patki PS. Speman®, a proprietary Ayurvedic formulation, reverses cyclophosphamide-induced oligospermia in rats. Central Asian Journal of Global Health. 2013; 2(1). http://dx.doi.org/10.5195/cajgh.2013.14
Domosławska A, Zduńczyk S, Janowski T. Improvement of sperm motility within one month under selenium and vitamin E supplementation in four infertile dogs with low selenium status. Journal of Veterinary Research. 2019;63(2):293-297. http://dx.doi.org/10.2478/jvetres-2019-0025
Rijsselaere T, Maes D, Hoflack G, De Kruif A, Van Soom A. Effect of body weight, age and breeding history on canine sperm quality parameters measured by the Hamilton‐Thorne analyser. Reproduction in domestic animals. 2007;42(2):143-148. http://dx.doi.org/10.1111/j.1439-0531.2006.00743.x
Bidarimath M, Khalaj K, Kridli RT, Kan FW, Koti M, Tayade C. Extracellular vesicle mediated intercellular communication at the porcine maternal-fetal interface: A new paradigm for conceptus-endometrial cross-talk. Scientific reports. 2017;7(1):40476. http://dx.doi.org/10.1038/srep40476
Barranco I, Padilla L, Parrilla I, Álvarez-Barrientos A, Pérez-Patiño C, Peña FJ, Martínez EA, Rodriguez-Martínez H, Roca J. Extracellular vesicles isolated from porcine seminal plasma exhibit different tetraspanin expression profiles. Scientific reports. 2019; 9(1):11584. https://www.nature.com/articles/s41598-019-48095-3
Heijnen HF, Schiel AE, Fijnheer R, Geuze HJ, Sixma JJ. Activated Platelets Release Two Types of Membrane Vesicles: Microvesicles by Surface Shedding and Exosomes Derived From Exocytosis of Multivesicular Bodies and-Granules. Blood, The Journal of the American Society of Hematology. 1999; 94(11):3791-3799. http://dx.doi.org/10.1182/blood.V94.11.3791
El Andaloussi S, Mäger I, Breakefield XO, Wood MJ. Extracellular vesicles: biology and emerging therapeutic opportunities. Nature reviews Drug discovery. 2013; 12(5):347-357. http://dx.doi.org/10.1038/nrd3978
Cocucci E, Meldolesi J. Ectosomes and exosomes: shedding the confusion between extracellular vesicles. Trends in cell biology. 2015; 25(6):364-372. http://dx.doi.org/10.1016/j.tcb.2015.01.004
Bahr M, Amer M, Abo-El-Sooud K, Kamel Sm FM, Abdallah A, Shamaa A, El-Tookhy O. Effect of topical application of lyophilized xenogenous mesenchymal stem cell-derived extracellular vesicles on central and peripheral corneal ulcers healing in rabbits. Adv Anim Vet Sci. 2023;11(1):132-140. http://dx.doi.org/10.17582/journal.aavs/2023/11.1.132.140
Théry C, Boussac M, Véron P, Ricciardi-Castagnoli P, Raposo G, Garin J, Amigorena S. Proteomic analysis of dendritic cell-derived exosomes: a secreted subcellular compartment distinct from apoptotic vesicles. The Journal of Immunology. 2001;166(12):7309-7318. http://dx.doi.org/10.4049/jimmunol.166.12.7309
Morello M, Minciacchi V, de Candia P, Yang J, Posadas E, Kim H, Griffiths D, Bhowmick N, Chung L, Gandellini P. Large oncosomes mediate intercellular transfer of functional microRNA. Cell cycle. 2013;12(22):3526-3536. http://dx.doi.org/10.4161/cc.26539
Anand S, Samuel M, Kumar S, Mathivanan S. Ticket to a bubble ride: Cargo sorting into exosomes and extracellular vesicles. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics. 2019;1867(12):140203. http://dx.doi.org/10.1016/j.bbapap.2019.02.005
Doyle LM, Wang MZ. Overview of Extracellular Vesicles, Their Origin, Composition, Purpose, and Methods for Exosome Isolation and Analysis. Cells. 2019; 8(7):727. http://dx.doi.org/10.3390/cells8070727
Bhatta B, Cooks T. Reshaping the tumor microenvironment: extracellular vesicles as messengers of cancer cells. Carcinogenesis. 2020; 41(11):1461-1470. http://dx.doi.org/10.1093/carcin/bgaa107
Hu W, Liu C, Bi Z-Y, Zhou Q, Zhang H, Li L-L, Zhang J, Zhu W, Song Y-Y-Y, Zhang F. Comprehensive landscape of extracellular vesicle-derived RNAs in cancer initiation, progression, metastasis and cancer immunology. Molecular Cancer. 2020; 19:1-23. https://link.springer.com/article/10.1186/s12943-020-01199-1
Yáñez-Mó M, Siljander PR-M, Andreu Z, Bedina Zavec A, Borràs FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J. Biological properties of extracellular vesicles and their physiological functions. Journal of extracellular vesicles. 2015; 4(1):27066. http://dx.doi.org/10.3402/jev.v4.27066
Royo F, Théry C, Falcón-Pérez JM, Nieuwland R, Witwer KW. Methods for separation and characterization of extracellular vesicles: results of a worldwide survey performed by the ISEV rigor and standardization subcommittee. Cells. 2020; 9(9):1955. http://dx.doi.org/10.3390/cells9091955
Kupcova Skalnikova H, Bohuslavova B, Turnovcova K, Juhasova J, Juhas S, Rodinova M, Vodicka P. Isolation and characterization of small extracellular vesicles from porcine blood plasma, cerebrospinal fluid, and seminal plasma. Proteomes. 2019; 7(2):17. http://dx.doi.org/10.3390/proteomes7020017
Roca J, Rodriguez-Martinez H, Padilla L, Lucas X, Barranco I. Extracellular vesicles in seminal fluid and effects on male reproduction. An overview in farm animals and pets. Animal Reproduction Science. 2022; 246:106853. http://dx.doi.org/10.1016/j.anireprosci.2021.106853
Candenas L, Chianese R. Exosome composition and seminal plasma proteome: A promising source of biomarkers of male infertility. International journal of molecular sciences. 2020;21(19):7022. https://doi.org/10.3390/ijms21197022
Ronquist G. Prostasomes are mediators of intercellular communication: from basic research to clinical implications. Journal of internal medicine. 2012; 271(4):400-413. http://dx.doi.org/10.1111/j.1365-2796.2011.02487.x
Deregibus MC, Cantaluppi V, Calogero R, Lo Iacono M, Tetta C, Biancone L, Bruno S, Bussolati B, Camussi G. Endothelial progenitor cell–derived microvesicles activate an angiogenic program in endothelial cells by a horizontal transfer of mRNA. Blood, The Journal of the American Society of Hematology. 2007; 110(7):2440-2448. http://dx.doi.org/10.1182/blood-2007-03-078709
Herrera MB, Fonsato V, Gatti S, Deregibus MC, Sordi A, Cantarella D, Calogero R, Bussolati B, Tetta C, Camussi G. Human liver stem cell‐derived microvesicles accelerate hepatic regeneration in hepatectomized rats. Journal of cellular and molecular medicine. 2010;14(6b):1605-1618. http://dx.doi.org/10.1111/j.1582-4934.2009.00860.x
Raposo G, Nijman HW, Stoorvogel W, Liejendekker R, Harding CV, Melief C, Geuze HJ. B lymphocytes secrete antigen-presenting vesicles. The Journal of experimental medicine. 1996;183(3):1161-1172. http://dx.doi.org/10.1084/jem.183.3.1161
Ratajczak J, Miekus K, Kucia M, Zhang J, Reca R, Dvorak P, Ratajczak M. Embryonic stem cell-derived microvesicles reprogram hematopoietic progenitors: evidence for horizontal transfer of mRNA and protein delivery. Leukemia. 2006; 20(5):847-856. http://dx.doi.org/10.1038/sj.leu.2404132
Surai P, Noble R, Sparks N, Speake B. Effect of long-term supplementation with arachidonic or docosahexaenoic acids on sperm production in the broiler chicken. Journal of reproduction and fertility. 2000;120(2):257-264. http://dx.doi.org/10.1530/reprod/120.2.257
Rooke J, Shao C, Speake B. Effects of feeding tuna oil on the lipid composition of pig spermatozoa and in vitro characteristics of semen. REPRODUCTION-CAMBRIDGE. 2001;121(2):315-322. http://dx.doi.org/10.1530/rep.0.1210315
Wathes DC, Abayasekara DRE, Aitken RJ. Polyunsaturated fatty acids in male and female reproduction. Biology of reproduction. 2007;77(2):190-201. https://doi.org/10.1095/biolreprod.107.060558
Safarinejad MR, Safarinejad S. The roles of omega-3 and omega-6 fatty acids in idiopathic male infertility. Asian journal of andrology. 2012; 14(4):514. http://dx.doi.org/10.1038/aja.2012.46
Domosławska A, Zduńczyk S, Niżański W, Jurczak A, Janowski T. Effect of selenium and vitamin E supplementation on semen quality in dogs with lowered fertility. Journal of Veterinary Research. 2015;59(1):85-90. http://dx.doi.org/10.1515/bvip-2015-0013
Kirchhoff K, Failing K, Goericke‐Pesch S. Effect of dietary vitamin E and selenium supplementation on semen quality in Cairn Terriers with normospermia. Reproduction in Domestic Animals. 2017; 52(6):945-952. http://dx.doi.org/10.1111/rda.13000
Alonge S, Melandri M, Aiudi G, Lacalandra GM. Advances in prostatic diagnostics in dogs. The role of canine prostatic specific esterase in the early diagnosis of prostatic disorders. Topics in companion animal medicine. 2018; 33(4):105-108. http://dx.doi.org/10.1053/j.tcam.2018.09.002
Alonge S, Melandri M, Leoci R, Lacalandra GM, Aiudi G. Ejaculation effect on blood testosterone and prostatic Pulsed‐Wave Doppler ultrasound in dogs. Reproduction in domestic animals. 2018;53:70-73. http://dx.doi.org/10.1111/rda.13277
de Souza MB, England GC, Mota Filho AC, Ackermann CL, Sousa CVS, de Carvalho GG, Silva HVR, Pinto JN, Linhares JCS, Oba E. Semen quality, testicular B-mode and Doppler ultrasound, and serum testosterone concentrations in dogs with established infertility. Theriogenology. 2015; 84(5):805-810. http://dx.doi.org/10.1016/j.theriogenology.2015.05.015
Abdelnaby EA, Emam IA, El-Sherbiny HR, Fadl AM. The effects of aging and gestational month on uteroplacental vascular perfusion, and umbilical artery hemodynamics in pregnant jennies. BMC Veterinary Research. 2022; 18(1):404. http://dx.doi.org/10.1186/s12917-022-03499-8
Maher M, Farghali H, Elsayed A, Emam I, Abdelnaby E, Reem R. A potential use of Doppler sonography for evaluating normal hemodynamic values of the hepatic, pancreatic and splenic vessels in domestic rabbits. Adv Anim Vet Sci. 2020; 8(5):506e518. http://dx.doi.org/10.17582/journal.aavs/2020/8.5.506.518
Abdelkhalek KG, Badawy AB, Abdelnaby EA, Fathi M. Relationship Between Testicular Morphometry, Vascular Waveform Pattern, Semen Picture, and Specific Serum Testosterone Levels in Baladi bucks Approaching Puberty. Journal of Advanced Veterinary Research. 2022; 12(5):573-581. https://www.advetresearch.com/index.php/AVR/article/view/1064
El-Sherbiny HR, Abdelnaby EA, Samir H, Fathi M. Addition of autologous platelet rich plasma to semen extender enhances cryotolerance and fertilizing capacity of buffalo bull spermatozoa. Theriogenology. 2022;194:104-109. http://dx.doi.org/10.1016/j.theriogenology.2022.09.029
Johnston, S.D., Kustritz, M.V.R. , Olson, P.N.S. 2001. Semen collection, evaluation, and preservation. In Canine and Feline Theriogenology. W.B. Saunders, Philadelphia, pp 287-306. https://espace.library.uq.edu.au/view/UQ:401893
Spruijt A, Kooistra H, Oei C, Vinke C, Schaefers‐Okkens A, De Gier J. The function of the pituitary‐testicular axis in dogs prior to and following surgical or chemical castration with the GnRH‐agonist deslorelin. Reproduction in Domestic Animals. 2023;58(1):97-108. http://dx.doi.org/10.1111/rda.14266
Domosławska A, Zdunczyk S. Clinical and spermatological findings in male dogs with acquired infertility: a retrospective analysis. Andrologia. 2020;52(11):e13802. http://dx.doi.org/10.1111/and.13802
Taya K. Radioimmunoassay for progesterone, testosterone and estradiol-17beta using^< 125> I-iodohistamine radioligands. Jpn J Anim Reprod. 1985; 31:186-197. http://dx.doi.org/10.1262/jrd1977.31.186
Ciribé F, Panzarella R, Pisu MC, Di Cerbo A, Guidetti G, Canello S. Hypospermia improvement in dogs fed on a nutraceutical diet. The Scientific World Journal. 2018; 2018(1):9520204. http://dx.doi.org/10.1155/2018/9520204
Schäfer-Somi S. New approaches to semen improvement in dogs. Rev bras reprod anim. 2015:141-145. https://pesquisa.bvsalud.org/portal/resource/pt/vti-12843
Risso A, Pellegrino FJ, Relling AE, Corrada Y. Effect of long-term fish oil supplementation on semen quality and serum testosterone concentrations in male dogs. International Journal of Fertility & Sterility. 2016;10(2):223. http://dx.doi.org/10.22074/ijfs.2016.4913
Soares JM, Avelar GF, França LR. The seminiferous epithelium cycle and its duration in different breeds of dog (Canis familiaris). Journal of anatomy. 2009; 215(4):462-471. http://dx.doi.org/10.1111/j.1469-7580.2009.01122.x
Amann R, Hammerstedt R, Veeramachaneni D. The epididymis and sperm maturation: a perspective. Reproduction, fertility and development. 1993; 5(4):361-381. http://dx.doi.org/10.1071/RD9930361
Girouard J, Frenette G, Sullivan R. Comparative proteome and lipid profiles of bovine epididymosomes collected in the intraluminal compartment of the caput and cauda epididymidis. International journal of andrology. 2011;34(5pt2):e475-e486. http://dx.doi.org/10.1111/j.1365-2605.2011.01203.x
Tarazona R, Delgado E, Guarnizo M, Roncero R, Morgado S, Sanchez-Correa B, Gordillo J, Dejulian J, Casado J. Human prostasomes express CD48 and interfere with NK cell function. Immunobiology. 2011;216(1-2):41-46. http://dx.doi.org/10.1016/j.imbio.2010.03.002
Simon AL, Kiehl M, Fischer E, Proctor JG, Bush MR, Givens C, Rabinowitz M, Demko ZP. Pregnancy outcomes from more than 1,800 in vitro fertilization cycles with the use of 24-chromosome single-nucleotide polymorphism–based preimplantation genetic testing for aneuploidy. Fertility and sterility. 2018; 110(1):113-121. http://dx.doi.org/10.1016/j.fertnstert.2018.03.026
Nixon B, De Iuliis GN, Hart HM, Zhou W, Mathe A, Bernstein IR, Anderson AL, Stanger SJ, Skerrett-Byrne DA, Jamaluddin MFB. Proteomic profiling of mouse epididymosomes reveals their contributions to post-testicular sperm maturation. Molecular & Cellular Proteomics. 2019;18:S91-S108. http://dx.doi.org/10.1074/mcp.RA118.000946
Abdelnaby EA, Emam IA, Fadl AM. Assessment of the accuracy of testicular dysfunction detection in male donkey (Equus asinus) with the aid of colour‐spectral Doppler in relation to plasma testosterone and serum nitric oxide levels. Reproduction in Domestic Animals. 2021;56(5):764-774. http://dx.doi.org/10.1111/rda.13916
El-Sherbiny HR, El-Shalofy AS, Samir H. Exogenous L-carnitine administration ameliorates the adverse effects of heat stress on testicular hemodynamics, echotexture, and total antioxidant capacity in rams. Frontiers in veterinary science. 2022;9:860771. http://dx.doi.org/10.3389/fvets.2022.860771
Jegou B, Sharpe R. Paracrine mechanisms in testicular control. Molecular biology of the male reproductive system. 1993:271-310. https://cir.nii.ac.jp/crid/1360011146211050880
Salama A, Abdelnaby EA, Emam IA, Fathi M. Single melatonin injection enhances the testicular artery hemodynamic, reproductive hormones, and semen parameters in German shepherd dogs. BMC veterinary research.2022;18(1):403. http://dx.doi.org/10.1186/s12917-022-03487-y
El-Seadawy IE, Kotp MS, El-Maaty AMA, Fadl AM, El-Sherbiny HR, Abdelnaby EA. The impact of varying doses of moringa leaf methanolic extract supplementation in the cryopreservation media on sperm quality, oxidants, and antioxidant capacity of frozen-thawed ram sperm. Tropical Animal Health and Production. 2022;54(6):344. http://dx.doi.org/10.1007/s11250-022-03344-y

No competing interests reported.

Download PDF

Reviewers agreed at journal
09 Sep, 2024
Reviews received at journal
01 Sep, 2024
Reviewers agreed at journal
22 Aug, 2024
Reviewers invited by journal
10 Aug, 2024
Editor invited by journal
07 Aug, 2024
Editor assigned by journal
05 Aug, 2024
Submission checks completed at journal
05 Aug, 2024
First submitted to journal
05 Aug, 2024

You are reading this latest preprint version

Therapeutic effect of single intra-testicular injection of lyophilized xenogenous and allogeneic mesenchymal stem cell-derived extracellular vesicles on oligozoospermia in male dogs.

Status:

Version 1

Abstract

Figures

1. Introduction

2. Material and method

2.1. Animals and management

2.2. Experimental design

2.3. Source of lyophilized canine and human MSCs-derived EVs

2.4. Single intra-testicular injection of lyophilized canine and human mesenchymal stem cell-derived extracellular vesicles

2.5. Ultrasonographic evaluation

2.6. Measurement of the testicular artery Doppler parameters

2.7. Semen collection and evaluation

2.8. Blood sampling and hormonal assaying

2.9. Statistical Analyses

3. Results

3.1. B-mode ultrasonic findings of volume of dog testes

3.2. Testicular hemodynamics

3.3. Semen analysis

3.4. Serum testosterone and NO levels.

4. Discussion

5. Conclusion

Declarations

References

Additional Declarations

Status:

Version 1