Microsurgical and Percutaneous Epididymal Sperm Aspiration for Sperm Collection from Live Mice

Abstract

Spermatozoa for in vitro fertilization of mouse oocytes and other methods of assisted reproduction typically are collected from the cauda epididymis of euthanized male mice. As an alternative to this terminal protocol, we developed and examined 2 methods for collecting sperm from anesthetized male mice without decreasing subsequent fertility: microsurgical epididymal sperm aspiration and, as a refinement, percutaneous epididymal sperm aspiration. Collected sperm was evaluated in terms of motility, concentration and in vitro fertilization ability. After recovery, both treated and untreated control male mice underwent in vivo fertility testing and subsequent histologic analysis of the treated male reproductive tract (epididymis and testis). In vitro fertilization using sperm recovered by the 2 collection methods was successfully achieved in all cases. The in vivo fertility test and the histologic analysis revealed no impairment of fertility and no permanent histologic alteration in the treated mice. Therefore, we recommend both techniques as simple and effective methods for recovering high-quality epididymal mouse sperm without having to euthanize fertile male mice.

The collection of mouse spermatozoa is a crucial technique for several methods of assisted reproduction. In vitro fertilization (IVF) of mouse oocytes is routinely used to overcome fertility problems, to rederive a mutant line, and to speed the expansion of a line.^14,15 Cryopreservation of sperm cells is currently the fastest and simplest method for storing unique and valuable mouse strains. Furthermore, sperm can be genotyped by PCR to determine whether a founder male of a transgenic line will transmit an induced mutation through the germline to the next generation.²⁴

The standard procedure for sperm collection is to euthanize the male mouse, dissect the vasa deferentia and caudae epididymides, strip the sperm from the vas deferens by using forceps or cannula, and then cut the caudae epididymidis to let the spermatozoa swim out.^14,15 This procedure yields the maximal number of mature spermatozoa from the mouse, but it is a terminal procedure and therefore can be risky when the available number of male mice is limited. As a compromise, one epididymis can be removed surgically and the other left to maintain in vivo fertility. However, this alternative can be performed only once in any male mouse.

For some applications that require less sperm, such as collecting sperm for IVF or genotyping to confirm the germline transmission of a mutation, the yield of sperm from the terminal procedure is much greater than is required. Often, it is more advantageous to keep the male mouse alive without any impairment of its fertility. Consequently, there is a high demand for a method of sperm collection that avoids euthanizing the sperm donor.

In the literature, there are 3 main methods of nonterminal sperm collection: electroejaculation; the injection of drugs; and flushing the uterus of females previously mated with the target male. Because all 3 of these methods are suitable for special experimental situations only, they have not found their way into routine practice. Electroejaculation has been applied successfully in larger domestic, farm, and zoo animals^4,9 and in humans such as men with erectile dysfunction or spinal cord injury.^10,20 The application of electroejaculation in mice has been described as early as the 1950s,^18,19 but this method is not routinely used because it requires specialized equipment (rectal probes, electrostimulator) and technical skills that are usually not present in a transgenic or cryopreservation lab. The efficiency (defined as percentage of successful ejaculations) in these early studies was high (80% to 96%), but the mortality of treated male mice varied between 0.5% and 22%,^18,19 which, in our opinion, was due to the high voltages (80 to 100 V) used. A more recent study using a computer-driven signal generator and much lower voltages (0.5 to 3 V) improved the collection efficiency and reduced mortality.²³

The second method, the pharmacologic induction of ejaculation, was used in the early days of reproductive pharmacology. In mice, ejaculation is facilitated by parasympathomimetic drugs (cholinesters, pilocarpine, physostigmine, prostigmine), and inhibited by parasympatholytic drugs (atropine).¹¹ The pernoston–yohimbine test was used as bioassay of male sex hormones.¹² The drawbacks of these methods included undesirable side effects and inconsistent success rates.²

The third method for mouse sperm collection is mating the sperm donors with female mice, euthanizing those with vaginal plugs, and flushing the sperm from the female genital tract into a culture dish.²⁶ However, this method can result in an increased susceptibility to DNA damage, probably due to enhanced nuclease activity in capacitated sperm which represent the majority of an ejaculate retrieved from the uterus.²⁶

In the present study, we describe 2 minimally invasive methods that allow for the collection of sperm from live male mice and do not cause more than mild or moderate pain and distress in the sperm donors. Specifically, we collected sperm by microsurgical and percutaneous epididymal sperm aspiration, both of which methods are used in human infertility treatments. Microsurgical epididymal sperm aspiration (MESA; also called microepididymal sperm aspiration) refers to the retrieval of sperm-containing fluid from optimal areas of the epididymis from patients with nonreconstructable obstructive azoospermia. In human assisted reproduction, the retrieved sperm are subsequently used for intracytoplasmic sperm injection to induce fertilization and pregnancy.¹⁷ It is viewed as the gold standard approach because it yields millions of high-quality, motile sperm with minimal specimen contamination.³ A second minimally invasive, safe and effective method is percutaneous epididymal sperm aspiration (PESA). PESA with subsequent intracytoplasmic sperm injection has been established in humans and yields results comparable to intracytoplasmic sperm injection using ejaculated spermatozoa.¹³

A method comparable with MESA has recently been applied in C57BL/6J mice.⁶ After scrotal access, one testis was exteriorized, and the cauda epididymis was clamped and punctured with a 32-gauge (0.202-mm) hypodermic needle. The epididymal sperm drop, appearing due to the applied pressure, was collected with capillary tubing (internal diameter, 0.58 mm) attached to a mouth pipette and used for IVF, with a mean fertilization rate of 56.7%.⁶

For our experiments, we compared the effectiveness of scrotal access and collection of sperm by direct puncture of the epididymis (MESA) with that of using a hypodermic needle to puncture the epididymis percutaneously, without opening the scrotum (PESA). Sperm obtained by these methods was used for the analysis of sperm quality and IVF. Furthermore, we tested whether either procedure impaired in vivo fertility, that is, the ability of the male to perform the act of sexual intercourse and deliver sperm that resulted in pregnancy. To this end, either MESA or PESA was performed on both testes, and after a recovery period of 2 weeks, the male mice were mated with female mice and the number of resulting offspring recorded. After a 5-week mating period, the testes and epididymides of the MESA- and PESA-treated male mice were examined histologically to reveal any morphologic alterations.

Materials and Methods

Animals.

Male (age, 10 wk) and female (age, 7 wk) B6D2F1 hybrid mice (C57BL/6NCrl × DBA/2NCrl) were purchased from Charles River Laboratories (Sulzfeld, Germany). Each male mouse was randomly assigned to a MESA, PESA, or untreated control group, and female mice were allocated to an IVF oocyte-donor or mating group. All mice had 7 to 14 d of adaption after arrival. CD1 female mice (age, 8 to 12 wk) of SPF quality, according to FELASA recommendations,⁸ and bred at our facility served as embryo-transfer recipients, and vasectomized CD1 male mice (age, 2 to 12 mo) from the same source were used to induce pseudopregnancy. Mice were housed in Macrolon cages (Tecniplast, Buguggiate, Italy) under standard laboratory conditions (room temperature, 21 ± 1 °C [mean ± SEM]; relative humidity, 40% to 55%; 12:12-h photoperiod) and supplied free choice with a standard breeding diet (V1126, Ssniff Spezialitäten, Soest, Germany) and tap water. The present study was discussed and approved by the institutional ethics committee of the University of Veterinary Medicine Vienna, and an animal experiment license was granted under BMWF-68.205/0137-II/3b/2012 (Austrian Federal Ministry of Science and Research).

Preparation of oocyte donors for IVF.

Three days before the planned sperm collection and IVF date, the oocyte donors were superovulated with an intraperitoneal injection of 5 IU pregnant mare serum gonadotropin (Folligon, Intervet, Vienna, Austria) at 1600. Two days later, 5 IU human chorionic gonadotropin (Chorulon, Intervet) was administered intraperitoneally at 1800.

MESA and PESA.

The surgery was performed in the morning (0700) of the planned IVF, about 13 h after the second hormone treatment of the oocyte donors. The sperm donor was anesthetized by injection of ketamine–xylazine (10 mg/100 g IP, Ketasol; 0.4 mg/100 g IP, Rompun; both from Graeub Veterinary Products, Bern, Switzerland). During anesthesia, eyes were covered with eye ointment (Oleovit, Fresenius Kabi, Graz, Austria). For postoperative analgesia, meloxicam (1 mg/kg; Metacam, Boehringer Ingelheim, Germany) was injected subcutaneously. Once a surgical plane of anesthesia was reached, the caudal abdomen was massaged gently downward to ensure that the testes were in the scrotum (Figure 1 A). The mouse was laid on its back, and the skin was disinfected with 70% ethanol and rinsed with distilled water. For a detailed view of the surgical area, a stereomicroscope (MZ125, Leica, Wetzlar, Germany) with a 12.5:1 zoom was used. By using scissors, a sagittal cut of about 10 mm was made in the skin at the bottom of the scrotum (Figure 1 B). The 2 caudae epididymides were easily visible through the intact tunica vaginalis. One technician used curved forces to fix a cauda epididymis and applied gentle pressure; a second person used a 30-gauge, 0.5-in. (0.255 mm × 12.7 mm) cannula on an insulin syringe (Omnican 50, B Braun, Melsungen, Germany) and prefilled with 0.05 mL of TYH medium (Figure 2) to puncture the tissue (Figure 1 C). After penetration of the cauda epididymis, negative pressure was applied on the syringe; successful aspiration was confirmed by increased turbidity of the media in the syringe. The contents of the syringe were expelled into a culture dish and checked for the presence of motile sperm. When sufficient (according to subjective evaluation) motile sperm was present, the aspirate was transferred into a 100-µL drop of TYH containing methyl-β-cyclodextrin in a 35-mm culture dish covered with mineral oil and incubated for 30 min at 37 °C and 5% CO₂ before sperm quality evaluation and IVF. When the aspirate contained insufficient motile spermatozoa, the puncturing procedure was repeated at most 2 times (for a maximum of 3 attempts) to limit the pain and distress of the mouse. The contralateral cauda epididymis was sampled in the same manner. The skin incision was closed with simple interrupted stitches of absorbable suture.

Figure 1.

Sperm aspiration by MESA and PESA techniques. (A) Gentle pressure is applied to the caudal region of the abdomen to transfer the testes into the scrotum. (B) MESA is performed after a sagittal cut to the skin, about 10 mm long, at the bottom of the scrotum. Gentle pressure is applied to one side of the scrotum. The right cauda epididymidis is visible through the tunica vaginalis. (C) The right cauda epididymidis is fixed with curved forceps by applying gentle pressure and then punctured through the tunica vaginalis by using an insulin syringe. (D) PESA of the left cauda epididymidis. Both caudae were fixed with curved forceps by applying gentle pressure; they are readily apparent at the bottom of the scrotum. The cauda is punctured through the skin, and sperm cells are aspirated into the syringe by applying negative pressure.

Figure 2.

Composition of TYH preincubation medium containing methyl-β-cyclodextrin and HTF fertilization medium containing l-glutamine.

For PESA, the anesthesia, analgesia, and preparation of the male mouse and scrotal area were performed as described for MESA. Because the shape of the caudae epididymides is visible at the bottom of the scrotum, one technician fixed the cauda by using curved forceps to apply gentle pressure. A second person pierced one cauda epididymis through the skin and aspirated the sperm by applying negative pressure (Figure 1 D). The treatment of the aspirate was the same as described for MESA. Each epididymis was punctured maximum 3 times.

At the same time as MESA or PESA procedures were performed, a B6D2F1 control male mouse was anesthetized for every sperm donor mouse used. Dosages, analgesia, and eye protection were the same as for the treated mice, but the control mice were otherwise left unmanipulated.

Examination of sperm quality.

After 30 min of incubation, the motility of the spermatozoa in the preincubation dish containing TYH medium was assessed subjectively. The percentage of motile and progressive sperm was graded, with 2+ signifying greater than 80% motile; +, greater than 60% to 80% motile; ±, greater than 40% to 60% motile; and –, less than 40% motile spermatozoa. Sperm concentration was determined by using a Neubauer counting chamber.²⁵

IVF.

The IVF procedure included a sperm preincubation phase in TYH medium with methyl-β-cyclodextrin and the addition of L-glutamine to the fertilization medium.^{1,5,15,21,22,27} Dishes for sperm preincubation (100 µL TYH plus methyl-β-cyclodextrin under mineral oil) were prepared in the afternoon before IVF, and the fertilization dish (200 µL HTF plus L-glutamine under mineral oil; Figure 2) was prepared immediately before IVF.

For IVF, the aspirated sperm was added to a 100-µL drop of TYH medium plus methyl-β-cyclodextrin and incubated for 30 min at 37 °C and 5% CO₂ in air. During this incubation period, the superovulated female mice were euthanized by cervical dislocation, and their oviducts were dissected and transferred into the mineral oil of the fertilization dish. The swollen ampulla was opened, and the cumulus–oocyte complexes were drawn into the fertilization drop. After the incubation period, 10 µL of sperm was removed from the preincubation dish (attempting to capture the spermatozoa with higher motility from the periphery of the drop while avoiding dead sperm and debris) and added to the cumulus–oocyte complexes. After insemination, the final sperm concentration was approximately 1:20 of the concentration measured in the incubation dish. The fertilization dishes were incubated for 4 to 6 h, after which the morphologically intact, presumptive zygotes were washed through 4 drops (150 µL each) of HTF and left in the last drop for overnight culture. The next morning, the 2-cell rate was calculated as a percentage of the total number of oocytes in the IVF.

In vivo fertility test.

Two weeks after the MESA or PESA experiment, the treated and control male mice were each mated with 2 B6D2F1 female mice. Five weeks later, the number of litters and offspring were recorded, and the number of days between pairing and the birth of the first litter (that is, the mating period) for each male mouse was determined.

Histologic examination.

After the mating period, all treated and control male mice were euthanized by cervical dislocation. The testes and the epididymides were dissected and fixed in 4% paraformaldehyde in PBS. After paraffin embedding, serial sections of the epididymides were stained with hematoxylin and eosin for evaluation. Serial histologic sections were scanned with a slide scanner (ScanScope CS, Aperio, Vista, CA).

Statistical analysis.

The results (mean values) of the in vivo fertility test (mating period and litter size) were compared between MESA- or PESA-treated male mice and untreated control male mice by using a t test. A P value of less than 5% was considered as significant.

Results

We were able to collect motile sperm from all 11 B6D2F1 male mice by using both the MESA and the PESA procedure. The data for sperm motility, sperm concentration, number of IVF oocytes, number of 2-cell embryos, and the 2-cell rates are given in Table 1. Sperm concentration varied from 3.36 to 17.47 × 10⁶ spermatozoa/mL for MESA and from 0.30 to 24.45 × 10⁶ spermatozoa/mL for PESA. All IVF trials were successful; the mean 2-cell rate after IVF was 84.2% ± 23.8% for MESA and 84.3% ± 15.2% for PESA. In all cases, sperm motility was estimated as 60% to 80%.

Table 1.

Results from the sperm quality evaluation and in vitro fertilization experiments using sperm collected by microsurgical epididymal sperm aspiration (MESA) and percutaneous epididymal sperm aspiration (PESA)

		Sperm		IVF
	Experiment	Motility	Concentration (x 106/mL)	No. of oocytes	No. of 2-cell embryos	Rate (%) of 2-cell embryos
MESA
	1	+	3.82	62	26	41.9
	2	+	4.76	83	83	100.0
	3	+	17.47	84	80	95.2
	4	+	5.61	45	41	91.1
	5	+	3.36	200	185	92.5
	Mean ± 1 SD		7.0 ± 5.9	94.8 ± 61.0	83.0 ± 62.1	84.2 ± 23.8
PESA
	1	+	3.90	94	81	86.2
	2	+	3.37	52	51	98.1
	3	+	0.30	121	71	58.7
	4	+	0.93	185	138	74.6
	5	+	24.45	25	24	96.0
	6	+	12.25	69	64	92.8
	Mean ± 1 SD		7.5 ± 9.3	91.0 ± 56.8	71.5 ± 38.1	84.4 ± 15.2

By using MESA, motile sperm were collected from both epididymides of 4 of the 5 male mice treated. PESA also led to motile sperm from all treated males, however, only in two cases from both epididymides (Table 2).

Table 2.

Results for individual epididymides regarding sperm quality and in vitro fertilization after sperm collection by MESA and PESA

	Left epididymis					Right epididymis
	Sperm		IVF			Sperm		IVF
Experiment	Motilitya	Concentration (x106/mL)	No. of oocytes	No. of 2-cell embryos	% of 2-cell embryos	Motilitya	Concentration (x 106/mL)	No. of oocytes	No. of 2-cell embryos	% of 2-cell embryos
MESA
1	+	6.00	46	10	21.7	+	1.64	16	16	100.0
2	+	2.93	40	40	100.0	+	6.59	43	43	100.0
3	+	4.94	45	45	100.0	+	30.00	39	35	89.7
4	—	ND	ND	ND	ND	+	5.61	45	41	91.1
5	+	5.96	124	112	90.3	+	0.75	76	73	96.1
Mean ± 1 SD		3.44 ± 1.44	60.80 ± 40.25	41.40 ± 43.04	62.41 ± 37.79		7.55 ± 12.05	39.50 ± 21.42	34.67 ± 20.54	79.48 ±4.83
PESA
1	+	3.40	48	40	83.3	+	4.39	46	41	89.1
2	+	2.95	17	16	94.1	+	3.78	35	35	100.0
3	—	ND	ND	ND	ND	+	0.30	121	71	58.7
4	—	ND	ND	ND	ND	+	0.93	185	138	74.6
5	+	24.45	25	24	96.0	—	ND	ND	ND	ND
6	—	ND	ND	ND	ND	+	12.25	69	64	92.8
Mean ± 1 SD		10.27 ± 15.20	30.00 ± 5.66	26.67 ± 5.66	91.13 ± 1.34		4.33 ± 5.50	91.20 ± 65.39	69.80 ± 43.55	83.04 ± 18.60

ND, not done

^a+ indicates >60% to 80% of sperm were motile; – indicates <40% motile spermatozoa.

After mating, all MESA- and PESA-treated male mice were able to produce offspring. Neither the mean mating period nor the mean litter size differed significantly from that of the control in either treatment (Table 3).

Table 3.

Results from the in-vivo fertility test with males treated by microsurgical epididymal sperm aspiration (MESA) or untreated males, and males treated by percutaneous epididymal sperm aspiration (PESA)

	Untreated male mice		MESA-treated male mice		PESA-treated male mice
Experiment	Mating perioda	Litter size	Mating perioda	Litter size	Mating perioda	Litter size
1	19	9.5	19	6	22	8.5
2	23	7.5	22	8	20.5	10
3	20	9.5	21	8.5	32	10
4	25	9	25	10.5	27.5	9.5
5	21	9	22	8.5	20	11
6	not done	not done	not done	not done	20	9,5
Mean ± 1 SD	21.6 ± 2.41	8.9 ± 0.82	21.8 ± 2.17	8.3 ± 1.6	23.7 ± 4.98	9.8 ± 0.82

^aMating period was defined as the number of days between mating and the first litter. Differences were tested as not significant.

The histologic examination of serial sections did not reveal any signs of pathologic alterations or other differences between the MESA- or PESA-treated and control male mice (data not shown).

Discussion

The purpose of this study was to develop an improved method for sperm collection from live mice. Two methods were applied: MESA and—as a refinement—PESA. Both methods are used in human andrology for sub- and infertile men.^3,13,17 The collected sperm cells are usually combined with intracytoplasmic sperm injection to obtain fertilization and pregnancy. We collected sperm from healthy male mice by MESA and PESA. After assessing sperm quality, we performed IVF according to recently published, very effective methods.²² After general anesthesia of a male mouse, both procedures were performed by using a 30-gauge, 0.5-in. cannula. Even with such a small cannula, it is not possible to puncture individual tubules of the mouse epididymal duct due to its small diameter. This situation is in contrast to human MESA, in which glass capillaries are inserted into epididymal tubules to collect 10 to 20 µL sperm within 10 to 20 min.³ In human PESA, a 21-gauge butterfly needle is moved back and forth within the epididymis, while negative pressure is applied by using a 20-mL syringe, until columns of slightly opalescent fluid are seen rising in the needle tubing and a satisfactory volume of sperm can be aspirated.¹³ In a similar manner, for PESA in mice, we had to move the needle carefully back and forth within the cauda epididymis while applying negative pressure until sperm was aspirated, which sometimes could be seen as faint cloudiness within the TYH medium in the syringe.

By using MESA or PESA, we were able to collect sperm from all 11 male mice used in this study. All sperm samples collected by the 2 methods showed similar good motility but varied in concentration (0.3 to 17.47 × 10⁶ spermatozoa/mL). Regardless, all samples were successfully used in IVF and achieved mean fertilization rates of 84.2% (range, 41.9% to 100%) for MESA and 84.4% (58.7% to 98.1%) for PESA. In this study, both epididymides were punctured so that any possible deleterious effects of the MESA or PESA procedure on the subsequent fertility of the male mouse and the histology of the epididymis could be examined. For practical applications, puncturing only one epididymis usually yields sufficient sperm for a successful IVF.

To limit the pain and distress in the mice and to avoid any pathologic changes of the tissue, we terminated sperm aspirations per epididymis after the 3rd trial. However, this practice did not always yield sufficient sperm. We observed that the testes and consequently the epididymides are slightly asymmetric within the scrotum in most male mice. Therefore, one cauda epididymis is more prominent and easier to access than the other.

In the in vivo fertility experiment, we showed that the procedure had no negative affect on the ability of the males to produce offspring. There were no differences in mean mating period after treatment or in mean litter size between MESA- or PESA-treated and untreated control male mice. This corresponds to previous findings regarding a MESA-like surgery in C57BL/6J male mice, where no deterioration of fertility was seen after in vivo mating even after the surgery was performed twice in the same male mouse.⁶ Compared with the surgical method of the previous study,⁶ the MESA technique we used can be considered less invasive—and thus a refinement—because the scrotum was opened only until the cauda epididymis could be seen, without opening the tunica vaginalis and without dislocating the testis and epididymis outside of the scrotum. The PESA method is clearly a further refinement, given that the procedure is performed without opening of the scrotum.

After completion of the in vivo fertility tests, we histologically examined the male reproductive tracts of all MESA- and PESA-treated and control male mice, focusing on the area of the epididymis but also including the deferent duct and testis. No differences indicative of a permanent alteration of the reproductive tissue were noted between treated and untreated mice. These results provide additional confirmation the benign nature of the MESA and PESA techniques when applied in the described manner. However, our results contrast with the observed changes found by histomorphometric analysis of the cauda epididymis 60 d after MESA treatment by using 22-gauge needles in rats.¹⁶ Although we did not perform the procedure twice in the same animal (as described earlier⁶), we conclude from our findings that after a waiting period of at least 2 wk, MESA or PESA could be repeated successfully. If only one cauda epididymis is punctured initially, using the contralateral epididymis during a repeat procedure would be an adequate option.

Sperm obtained from MESA or PESA proved to be of sufficient quality for cryopreservation. However, whether the lower yield of sperm (as compared with other traditional methods) can notably decrease the success of a cryopreservation procedure remains to be determined. If MESA- or PESA-derived sperm can be reliably cryopreserved and recovered, it would considerably broaden the application of both methods for the distribution of genetically altered mice.

The advantages of the MESA and PESA techniques to collect sperm from male mice males as conducted in this study compared with methods described in the literature are clear: our technique is quite easy to perform and, in contrast to the electroejaculation method, does not require any equipment that is not routinely present in a mouse transgenic or cryopreservation lab. Another significant benefit of MESA or PESA is that the harvested sperm is of sufficient quality and purity to be used for standard IVF without the need for challenging procedures such as partial zona dissection or intracytoplasmic sperm injection. This situation is in contrast to the sperm samples of generally inferior quality that are obtained by electroejaculation (reduced sperm concentration and motility, contaminated with urine and secretions of the accessory glands).²³ Furthermore, semen tends to coagulate at the time of ejaculation. The coagulum formation remains a significant impediment to the collection and examination of sperm from the ejaculate and increases the risk of mortality due to retention of coagulated semen within the urethra or bladder, leading to lethal uremia.²³

Unlike the potential consequences of a pharmacologic induction of ejaculation,² we never observed any serious or lasting side effects of MESA or PESA in mice. The moderate postoperative pain can be alleviated by using analgesic drugs like meloxicam applied immediately after manipulation for 1 to 2 d postoperatively. Moreover, in contrast to immediate flushing of the uterine horns after mating,²⁶ MESA and PESA exclude the use of female mice, thus following the principle of reduction as prescribed by the directive 2010/63/EU (Article 4).⁷

In summary, we developed 2 refined methods for the collection of epididymal sperm from genetically unique or otherwise valuable male mice. The collected sperm is of sufficient quality (motility and concentration) for subsequent IVF and can be used for fertility phenotyping or genotyping, for example, of chimeras to evaluate germ-line transmission. When applying the procedures in practice, we suggest trying PESA first because it is less invasive, and when insufficient motile sperm are collected, then opening the scrotum surgically for MESA, where the cauda epididymis can be visualized and punctured.

The presented techniques are now used routinely in our lab and have been successfully applied to a variety of mouse strains, including inbred backgrounds such as C57BL/6NCrl and BALB/cAnNCrl, with comparable results to those from the hybrids we used in the current study (data not shown).